Dataset Information

Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii.

ABSTRACT: Background:Acinetobacter baumannii (A. baumannii) is one of the pivotal pathogens responsible for nosocomial infections, especially in patients with low immune response, and infection with carbapenem-resistant A. baumannii has been increasing in recent years. Rapid and accurate detection of carbapenem-resistance genes in A. baumannii could be of immense help to clinical staff. Methods:In this study, a 15-?L reaction system for recombinase polymerase amplification (RPA) was developed and tested. We collected 30 clinical isolates of A. baumannii from the Burn Institute of Southwest Hospital of Third Military Medical University (Army Medical University) for 6 months and tested antibiotic susceptibility using the VITEK 2 system. A. baumannii was detected based on the bla OXA-51 gene by PCR, qPCR and 15 ?L-RPA, respectively. Sensitivity and specificity were evaluated. In addition, PCR and 15 ?L-RPA data for detecting the carbapenem-resistance gene bla OXA-23 were comparatively assessed. Results:The detection limit of the bla OXA-51 gene by 15 ?L RPA was 2.86 CFU/ml, with sensitivity comparable to PCR and qPCR. No positive amplification signals were detected in non-Acinetobacter isolates, indicating high specificity. However, only 18 minutes were needed for the 15 ?L RPA assay. Furthermore, an antibiotic susceptibility test showed that up to 90% of A. baumannii strains were resistant to meropenem and imipenem; 15 ?L RPA data for detecting bla OXA-23 showed that only 10% (n?=?3) of A. baumannii isolates did not show positive amplification signals, and the other 90% of (n?=?27) isolates were positive, corroborating PCR results. Conclusion:We demonstrated that the new 15 ?L RPA assay for detecting bla OXA-23 in A. baumannii is faster and simpler than qPCR and PCR. It is a promising alternative molecular diagnostic tool for rapid and effective detection of A. baumannii and drug-resistance genes in the field and point-of-care testing.

SUBMITTER: Liu S

PROVIDER: S-EPMC7462923 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in <i>Acinetobacter baumannii</i>.

Liu Shuang S Huang Guangtao G Gong Yali Y Jin Xiaojun X Meng Yudan Y Peng Yizhi Y Zhao Junning J Li Xiaolu X Li Qin Q

Burns & trauma 20200902

<h4>Background</h4><i>Acinetobacter baumannii</i> (A. <i>baumannii</i>) is one of the pivotal pathogens responsible for nosocomial infections, especially in patients with low immune response, and infection with carbapenem-resistant <i>A. baumannii</i> has been increasing in recent years. Rapid and accurate detection of carbapenem-resistance genes in <i>A. baumannii</i> could be of immense help to clinical staff.<h4>Methods</h4>In this study, a 15-μL reaction system for recombinase polymerase amp ...[more]

PMID: 32905076

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Project description:Background: Acinetobacter calcoaceticus-baumannii (ACB) complex has emerged as an important nosocomial pathogen and is associated with life-threatening infections, especially among ICU patients, including neonates. Carbapenem resistance in Acinetobacter baumannii has emerged globally and is commonly mediated by bla OXA-23. Clinically significant infections with carbapenem-resistant Acinetobacter baumannii (CRAB) are a major concern since therapeutic options are limited and associated mortality is high. Early diagnosis of both the pathogen and resistance is important to initiate the optimal therapy and prevent selection of resistance. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the ACB complex and carbapenem resistance mediated by bla OXA-23. Methodology: Universal LAMP primers were designed for the detection of significant members of the ACB complex and carbapenem resistance targeting the ITS 16S-23S rRNA and bla OXA-23 gene respectively. The optimal conditions for the LAMP assay were standardized for each primer set using standard ATCC strains. The sensitivity of the LAMP assay was assessed based on the limit of detection (LOD) using different DNA concentrations and colony counts. The specificity of LAMP was determined using the non-ACB complex and non-Acinetobacter species. The results of the LAMP assay were compared with those of polymerase chain reaction (PCR). Results: The optimal temperature for the LAMP assay was 65°C, and the detection time varied with various primers designed. Using the ITS Ab1 primer, LODs of LAMP and PCR assays were 100 pg/μl and 1 ng/μl of DNA concentration and 104 cfu/ml and 108 cfu/ml of colony count, respectively. The LAMP assay was 10- and 104-fold more sensitive than PCR using DNA concentration and colony count, respectively. The LAMP assay was found to be specific for clinically important ACB complex species. Significance of the study: The LAMP assay can be applied for early detection of significant species of the ACB complex from clinical samples and their carbapenem-resistant variants. Depending on the emerging pathogen and locally prevalent resistance genes, the LAMP assay can be modified for detection of colonization or infection by various resistant bugs.

Project description:BackgroundDetection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.Methodology and significant findingsSpecies-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.ConclusionThe RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.

Project description:BackgroundWith an increase in the use of colistin methansulfonate (CMS) to treat carbapenem-resistant Acinetobacter baumannii infections, colistin resistance is emerging.MethodsPatients with infection or colonization due to colistin-resistant A. baumannii were identified at a hospital system in Pennsylvania. Clinical data were collected from electronic medical records. Susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were performed. To investigate the mechanism of colistin resistance, lipid A was subjected to matrix-assisted laser desorption/ionization mass spectrometry.ResultsTwenty patients with colistin-resistant A. baumannii were identified. Ventilator-associated pneumonia was the most common type of infection. Nineteen patients had received intravenous and/or inhaled CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection prior to identification of colistin-resistant isolates. The 30-day all-cause mortality rate was 30%. The treatment regimen for colistin-resistant A. baumannii infection associated with the lowest mortality rate was a combination of CMS, a carbapenem, and ampicillin-sulbactam. The colistin-susceptible and -resistant isolates from the same patients were highly related by PFGE, but isolates from different patients were not, suggesting evolution of resistance during CMS therapy. By MLST, all isolates belonged to the international clone II, the lineage that is epidemic worldwide. Phosphoethanolamine modification of lipid A was present in all colistin-resistant A. baumannii isolates.ConclusionsColistin-resistant A. baumannii occurred almost exclusively among patients who had received CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection. Lipid A modification by the addition of phosphoethanolamine accounted for colistin resistance. Susceptibility testing for colistin should be considered for A. baumannii identified from CMS-experienced patients.

Project description:Background and objectivesAcinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-detecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.Materials and methodsLAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the bla SHV-1 , bla PER-1 , bla TEM-1, AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic identification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilutions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.ResultsAmong one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL producers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were bla SHV-1 positive, (40%) bla PER-1, (85%) aac (6')-1, AMPC (67%), bla TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla SHV-1 positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla TEM-1 , and bla PER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.ConclusionWherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detection rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. baumannii in these clinical isolates showed frequent commonality of genotypes.

Project description:Gene amplification is believed to play an important role in antibiotic resistance but has been rarely documented in clinical settings because of its unstable nature. We report a rise in MICs from 0.5 to 16 ?g/ml in successive Acinetobacter baumannii isolated over 4 days from a patient being treated with tobramycin for an infection by multidrug-resistant A. baumannii, resulting in therapeutic failure. Isolates were characterized by whole-genome sequencing, real-time and reverse transcriptase PCR, and growth assays to determine the mechanism of tobramycin resistance and its fitness cost. Tobramycin resistance was associated with two amplification events of different chromosomal fragments containing the aphA1 aminoglycoside resistance gene part of transposon Tn6020. The first amplification event involved low amplification (6 to 10 copies) of a large DNA fragment that was unstable and conferred tobramycin MICs of ? 8 ?g/ml. The second event involved moderate (10 to 30 copies) or high (40 to 110 copies) amplification of Tn6020. High copy numbers were associated with tobramycin MICs of 16 ?g/ml, impaired fitness, and genetic instability, whereas lower copy numbers resulted in tobramycin MICs of ?8 ?g/ml and no fitness cost and were stably maintained in vitro. Exposure in vitro to tobramycin of the initial susceptible isolate and of the A. baumannii AB0057 reference strain led to similar aphA1 amplifications and elevated tobramycin MICs. To the best of our knowledge, this is the first report of in vivo development of antibiotic resistance secondary to gene amplifications resulting in therapy failure. IMPORTANCE A combination of whole-genome sequencing and mapping were used to detect an antibiotic resistance mechanism, gene amplification, which has been presumed for a long time to be of major importance but has rarely been reported in clinical settings because of its unstable nature. Two gene amplification events in a patient with an Acinetobacter baumannii infection treated with tobramycin were identified. One gene amplification event led to high levels of resistance and was rapidly reversible, while the second event led to low and more stable resistance since it incurred low fitness cost on the host. Gene amplification, with an associated rise in tobramycin MICs, could be readily reproduced in vitro from initially susceptible strains exposed to increasing concentrations of tobramycin, suggesting that gene amplification in A. baumannii may be a more common mechanism than currently believed. This report underscores the importance of rapid molecular techniques for surveillance of drug resistance.

Dataset Information

Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii.

Publications

Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in <i>Acinetobacter baumannii</i>.

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