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      Rapid detection of carbapenemase activity of Enterobacteriaceae isolated from positive blood cultures by MALDI-TOF MS

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          Abstract

          Background

          Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been proved to be a useful tool for identification of pathogens directly isolated from blood cultures in clinical microbiology laboratories. β-Lactam antibiotics are commonly used for treatment of bloodstream infections caused by Enterobacteriaceae strains, and carbapenem is the superlative class of β-lactam antibiotics. Since the carbapenem resistance rate of Enterobacteriaceae strains raised year by year, efficient detection of carbapenemase activity and timely delivery of carbapenem susceptibility reports of Enterobacteriaceae strains isolated from blood cultures is important for clinicians.

          Methods

          We used 64 simulated blood cultures to establish the method of MALDI-TOF MS based ertapenem hydrolysis assay. The cutoff value of logRQ calculated from the peaks intensity of ertapenem and its hydrolysate was first set to identify the strains with carbapenemase activity. Then, we detected and calculated the logRQ values of 385 Enterobacteriaceae strains from positive clinical blood cultures to distinguish the carbapenemase producers and noncarbapenemase producers.

          Results

          The mean logRQ value of 32 noncarbapenemase producers was − 0.85 ± 0.14 in simulated blood cultures, while the logRQ value of 32 carbapenemase producers was 0.87 ± 0.55. Thus, the cutoff value of logRQ was set at − 0.45 with sensitivity of 100% and specificity of 100%. In 385 clinical positive blood cultures, the logRQ values of all carbapenem-susceptible Enterobacteriaceae strains (81.3%, 313/385) were < − 0.45. Comparing with the detection of carbapenemase genes, carbapenem-resistant Enterobacteriaceae strains (18.7%, 72/385) were well distinguished by MALDI-TOF MS based ertapenem hydrolysis assay with a sensitivity of 92.5% and specificity of 100%.

          Conclusions

          Our data show that MALDI-TOF MS based ertapenem hydrolysis assay is a rapid and accurate method to detect carbapenemase activity of Enterobacteriaceae strains from positive blood cultures, and can be routinely performed in clinical microbiology laboratories.

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          Most cited references22

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          Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock.

          Our goal was to determine the impact of the initiation of inappropriate antimicrobial therapy on survival to hospital discharge of patients with septic shock. The appropriateness of initial antimicrobial therapy, the clinical infection site, and relevant pathogens were retrospectively determined for 5,715 patients with septic shock in three countries. Therapy with appropriate antimicrobial agents was initiated in 80.1% of cases. Overall, the survival rate was 43.7%. There were marked differences in the distribution of comorbidities, clinical infections, and pathogens in patients who received appropriate and inappropriate initial antimicrobial therapy (p < 0.0001 for each). The survival rates after appropriate and inappropriate initial therapy were 52.0% and 10.3%, respectively (odds ratio [OR], 9.45; 95% CI, 7.74 to 11.54; p < 0.0001). Similar differences in survival were seen in all major epidemiologic, clinical, and organism subgroups. The decrease in survival with inappropriate initial therapy ranged from 2.3-fold for pneumococcal infection to 17.6-fold with primary bacteremia. After adjustment for acute physiology and chronic health evaluation II score, comorbidities, hospital site, and other potential risk factors, the inappropriateness of initial antimicrobial therapy remained most highly associated with risk of death (OR, 8.99; 95% CI, 6.60 to 12.23). Inappropriate initial antimicrobial therapy for septic shock occurs in about 20% of patients and is associated with a fivefold reduction in survival. Efforts to increase the frequency of the appropriateness of initial antimicrobial therapy must be central to efforts to reduce the mortality of patients with septic shock.
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            Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014.

            With the aim of gathering temporal trends on bacterial epidemiology and resistance from multiple laboratories in China, the CHINET surveillance system was organized in 2005. Antimicrobial susceptibility testing was carried out according to a unified protocol using the Kirby-Bauer method or automated systems. Results were analyzed according to Clinical and Laboratory Standards Institute (CLSI) 2014 definitions. Between 2005 and 2014, the number of bacterial isolates ranged between 22,774 and 84,572 annually. Rates of extended-spectrum β-lactamase production among Escherichia coli isolates were stable, between 51.7 and 55.8%. Resistance of E. coli and Klebsiella pneumoniae to amikacin, ciprofloxacin, piperacillin/tazobactam and cefoperazone/sulbactam decreased with time. Carbapenem resistance among K. pneumoniae isolates increased from 2.4 to 13.4%. Resistance of Pseudomonas aeruginosa strains against all of antimicrobial agents tested including imipenem and meropenem decreased with time. On the contrary, resistance of Acinetobacter baumannii strains to carbapenems increased from 31 to 66.7%. A marked decrease of methicillin resistance from 69% in 2005 to 44.6% in 2014 was observed for Staphylococcus aureus. Carbapenem resistance rates in K. pneumoniae and A. baumannii in China are high. Our results indicate the importance of bacterial surveillance studies.
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              Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK.

              The aim of this study was to investigate relatedness and molecular mechanism(s) of ertapenem resistance in clinical isolates of Klebsiella spp. (n = 28) and Enterobacter spp. (n = 27) referred from multiple hospitals to the UK national reference laboratory. Investigations included genotyping by PFGE, resistance gene analysis by PCR and antimicrobial susceptibility testing with and without inhibitors of efflux and beta-lactamase activity. Outer membrane proteins (OMPs) were profiled by SDS-PAGE; porin genes were sequenced and their expression was examined by RT-PCR. The contribution of porin deficiency to resistance was investigated by restoring functional porin genes on plasmids. PFGE showed significant clonal diversity among ertapenem-resistant isolates, with only small clusters identified. SHV- and CTX-M-type extended-spectrum beta-lactamases were identified in the Klebsiella spp. isolates, whereas AmpC overexpression or KPC carbapenemase was detected in the Enterobacter cloacae isolates. SDS-PAGE showed that Klebsiella pneumoniae and Enterobacter aerogenes with high-level ertapenem resistance (MICs > or = 16 mg/L) consistently lacked both of the two major non-specific porins, whereas variable patterns of OmpC and OmpF were seen in E. cloacae with lower-level ertapenem resistance. Various point mutations or insertion sequences were identified as disrupting the porin-coding sequences, as well as mutations in the promoter region. Functional restoration of OmpK35 or OmpK36 in Klebsiella and OmpC or OmpF in Enterobacter spp. isolates significantly decreased the MICs of all carbapenems, but particularly of ertapenem. We found no evidence of efflux contributing to resistance. Ertapenem resistance was exclusively due to combinations of beta-lactamases with impermeability caused by loss of OMPs. Efflux was not implicated and there was no national spread of resistant clones.
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                Author and article information

                Contributors
                yujiajia0302@163.com
                ljxwwwyy@163.com
                liyuanrui729@126.com
                yujing02@xinhuamed.com.cn
                xinyimin163@126.com
                liuying01@xinhuamed.com.cn
                lisongshen@hotmail.com
                Journal
                Ann Clin Microbiol Antimicrob
                Ann. Clin. Microbiol. Antimicrob
                Annals of Clinical Microbiology and Antimicrobials
                BioMed Central (London )
                1476-0711
                18 May 2018
                18 May 2018
                2018
                : 17
                : 22
                Affiliations
                ISNI 0000 0004 0368 8293, GRID grid.16821.3c, Department of Clinical Laboratory, Xin Hua Hospital, , Shanghai Jiao Tong University School of Medicine, ; 1665 Kong Jiang Road, Yangpu District, Shanghai, 200092 China
                Article
                274
                10.1186/s12941-018-0274-9
                5960105
                29776363
                6c6b905c-0173-4ca2-ae80-6a7be66899f9
                © The Author(s) 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 30 August 2017
                : 5 May 2018
                Categories
                Research
                Custom metadata
                © The Author(s) 2018

                Infectious disease & Microbiology
                maldi-tof ms,blood culture,carbapenemase,enterobacteriaceae,hydrolysis

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