scholarly journals Development of Loop-Mediated Isothermal Amplification Assay for Detection of Clinically Significant Members of Acinetobacter calcoaceticus–baumannii Complex and Associated Carbapenem Resistance

2021 ◽  
Vol 8 ◽  
Author(s):  
Amit Sharma ◽  
Rajni Gaind
Keyword(s):  
Acinetobacter Baumannii ◽  
Lamp Assay ◽  
Carbapenem Resistance ◽  
Acinetobacter Calcoaceticus ◽  
Isothermal Amplification ◽  
Colony Count ◽  
Dna Concentration ◽  
Loop Mediated Isothermal Amplification ◽  
Carbapenem Resistant ◽  
Clinically Significant

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 blaOXA-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 blaOXA-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 blaOXA-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.

scholarly journals A multiplex loop-mediated isothermal amplification assay for rapid screening of Acinetobacter baumannii and D carbapenemase OXA-23 gene

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Rungong Yang ◽  
Honghong Zhang ◽  
Xiaoxia Li ◽  
Ling Ye ◽  
Meiliang Gong ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acinetobacter Baumannii ◽  
Resistance Gene ◽  
Traditional Method ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Rapid Screening ◽  
Reca Gene ◽  
Drug Resistance Gene ◽  
Loop Mediated Isothermal Amplification

Background: Acinetobacter baumannii is a health burden responsible for various nosocomial infections, and bacteremia in particular. The resistance of A. baumannii to most antibiotics including carbapenem has increased. OXA-23-producing A. baumannii is the chief source of nosocomial outbreaks with carbapenem-resistant A. baumannii. Successful antibiotic treatment relies on the accurate and rapid identification of infectious agents and drug resistance. Here, we describe a multiplex loop-mediated isothermal amplification (LAMP) assay for simultaneous and homogeneous identification for A. baumannii infection screening and drug-resistance gene detection. Methods: Four primer pairs were designed to amplify fragments of the recA gene of A. baumannii and the oxa-23 gene. The reaction with a 25 μl of final volume was performed at 63°C for 60 min. For comparative purposes, we used a traditional method of bacterial identification to evaluate assay efficacy. Results: The multiplex LAMP assay enables simultaneous and homogeneous detection of the recA gene of A. baumannii and the oxa-23 gene and requires less than 21 min with no pre-requisite for DNA purification prior to the amplification reaction. The detection is specific to A. baumannii, and the coincidence rate of the multiplex LAMP and the traditional method was 100%. Conclusions: Our data indicate that the multiplex LAMP assay is a rapid, sensitive, simultaneous and homogeneous method for screening of A. baumannii and its drug-resistance gene.

scholarly journals Evaluation of a Loop-Mediated Isothermal Amplification assay to detect carbapenemases from Acinetobacter spp. directly from bronchoalveolar lavage fluid

2020 ◽  
Author(s):  
J. Moreno-Morales ◽  
A. Vergara ◽  
T. Kostyanev ◽  
J. Rodriguez-Baño ◽  
H. Goossens ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Acinetobacter Baumannii ◽  
Nosocomial Infections ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Carbapenem Resistant ◽  
Amplification Assay ◽  
Acinetobacter Spp

AbstractCarbapenem-resistant Acinetobacter spp. mainly Acinetobacter baumannii are frequently causing nosocomial infections with high mortality. In this study, the efficacy of the Eazyplex® SuperBug Complete A system (Amplex Diagnostics GmbH, Gars-Bahnhof, Germany), based on loop-mediated isothermal amplification (LAMP), to detect the presence of carbapenemases in Acinetobacter spp. directly from bronchoalveolar lavage samples was assessed, detecting all tested carbapenemases in less than 30 minutes with a sensitivity of 103 CFU/ml.

scholarly journals Evaluation of a Loop-Mediated Isothermal Amplification-Based Methodology To Detect Carbapenemase Carriage in Acinetobacter Clinical Isolates

2014 ◽  
Vol 58 (12) ◽  
pp. 7538-7540 ◽  
Author(s):  
Andrea Vergara ◽  
Yuliya Zboromyrska ◽  
Noraida Mosqueda ◽  
María Isabel Morosini ◽  
Sergio García-Fernández ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Nosocomial Infections ◽  
Isothermal Amplification ◽  
Clinical Isolates ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
System A ◽  
Carbapenem Resistant ◽  
Different Types ◽  
Hydrolyzing Enzymes

ABSTRACTCarbapenem-resistantAcinetobacter baumanniiis a major source of nosocomial infections worldwide and is mainly associated with the acquisition of OXA-type carbapenemases and, to a lesser extent, metallo-β-lactamases (MBLs). In this study, 82 nonepidemiologically relatedAcinetobacterstrains carrying different types of OXA or MBL enzymes were tested using the Eazyplex system, a loop-mediated isothermal amplification (LAMP)-based method to rapidly detect carbapenemase carriage. The presence/absence of carbapenem-hydrolyzing enzymes was correctly determined for all isolates in <30 min.

scholarly journals Types and Prevalence of Carbapenem-Resistant Acinetobacter calcoaceticus-Acinetobacter baumannii Complex in Northern Taiwan

2013 ◽  
Vol 58 (1) ◽  
pp. 201-204 ◽  
Author(s):  
Wen-Shyang Hsieh ◽  
Nai-Yu Wang ◽  
Jou-An Feng ◽  
Li-Chuan Weng ◽  
Hsueh-Hsia Wu
Keyword(s):  
Acinetobacter Baumannii ◽  
Medical Center ◽  
Carbapenem Resistance ◽  
Acinetobacter Calcoaceticus ◽  
Pulsed Field Gel Electrophoresis ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Acinetobacter Baumannii Complex ◽  
High Level ◽  
Northern Taiwan

ABSTRACTThe frequency of the carbapenem-resistantAcinetobacter calcoaceticus-Acinetobacter baumannii(CRACB) complex increases annually in our hospitals. However, the types and prevalence of carbapenemases among isolates still remain unclear. In this study, we identified and collected 672 carbapenem-resistant isolates from a medical center in Northern Taiwan between April and December of 2010. There were 577 genospecies 2 (Acinetobacter baumannii), 79 genospecies 13TU, and 16 genospecies 3 isolates. The isolates had an acquiredblaOXA-24-like gene, which was confirmed by sequencing for the encoded OXA-72 carbapenemase, and were often associated with high-level carbapenem resistance. These CRACB complex isolates remained susceptible to colistin (100%). The genotyping of isolates was conducted using pulsed-field gel electrophoresis with ApaI digestion. In most clonally related groups, patients were from both branch hospitals. The results indicate that interhospital dissemination of clones occurred. This study provides updated data on the types and prevalence of the CRACB complex. In addition, it presents a warning on the emergence and spread of CRACB complex harboringblaOXA-24-like genes in northern Taiwan.

scholarly journals Evaluation of Clonality and Carbapenem Resistance Mechanisms among Acinetobacter baumannii-Acinetobacter calcoaceticus Complex and Enterobacteriaceae Isolates Collected in European and Mediterranean Countries and Detection of Two Novel β-Lactamases, GES-22 and VIM-35

2014 ◽  
Vol 58 (12) ◽  
pp. 7358-7366 ◽  
Author(s):  
Mariana Castanheira ◽  
Sarah E. Costello ◽  
Leah N. Woosley ◽  
Lalitagauri M. Deshpande ◽  
Todd A. Davies ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Carbapenem Resistance ◽  
Acinetobacter Calcoaceticus ◽  
Resistance Mechanisms ◽  
The Novel ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Mediterranean Countries ◽  
Class 1

ABSTRACTWe evaluated doripenem-resistantAcinetobacter baumannii-Acinetobacter calcoaceticuscomplex (ACB;n= 411) andEnterobacteriaceae(n= 92) isolates collected from patients from 14 European and Mediterranean countries during 2009 to 2011 for the presence of carbapenemase-encoding genes and clonality. Following susceptibility testing, carbapenem-resistant (doripenem MIC, >2 μg/ml) isolates were screened for carbapenemases. New β-lactamase genes were expressed in a common background and susceptibility was tested. Class 1 integrons were sequenced. Clonality was evaluated by pulsed-field gel electrophoresis and multilocus sequence typing (Pasteur scheme). Relative expression of β-lactam intrinsic resistance mechanisms was determined for carbapenemase-negativeEnterobacteriaceae. ACB andEnterobacteriaceaedisplayed 58.9 and 0.9% doripenem resistance, respectively.blaOXA-23,blaOXA-58, andblaOXA-24/OXA-40were detected among 277, 77, and 29 ACB, respectively (in 8, 6, and 5 countries). Ten Turkish isolates carriedblaGES-11orblaGES-22. GES-22 (G243A and M169L mutations in GES-1) had an extended-spectrum β-lactamase profile. A total of 33 clusters of ≥2 ACB isolates were observed, and 227 isolates belonged to sequence type 2/international clone II. Other international clones were limited to Turkey and Israel. Doripenem-resistantEnterobacteriaceaeincreased significantly (0.7 to 1.6%), and 15blaKPC-2- and 22blaKPC-3-carrying isolates, mostly belonging to clonal complexes 11 and 258, were observed.Enterobacteriaceaeisolates producing OXA-48 (n= 16; in Turkey and Italy), VIM-1 (n= 10; in Greece, Poland, and Spain), VIM-26 (n= 1; in Greece), and IMP-19, VIM-4, and the novel VIM-35 (n= 1 each from Poland) were detected. VIM-35 had one substitution compared to VIM-1 (A235T) and a similar susceptibility profile. One or more resistance mechanisms were identified in 4/6 carbapenemase-negativeEnterobacteriaceae. This broad evaluation confirms results from country-specific surveys and shows a highly diverse population of carbapenemase-producing ACB andEnterobacteriaceaein Europe and Mediterranean countries.

Acinetobacter calcoaceticus–Acinetobacter baumannii complex species in clinical specimens in Singapore

2011 ◽  
Vol 140 (3) ◽  
pp. 535-538 ◽  
Author(s):  
T. H. KOH ◽  
T. T. TAN ◽  
C. T. KHOO ◽  
S. Y. NG ◽  
T. Y. TAN ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Species Complex ◽  
Carbapenem Resistance ◽  
Acinetobacter Calcoaceticus ◽  
Complex Species ◽  
Clinical Specimens ◽  
Carbapenem Resistant ◽  
Acinetobacter Baumannii Complex ◽  
Acinetobacter Pittii ◽  
Resistant Strains

SUMMARYThis study was performed to determine the prevalence, distribution of specimen sources, and antimicrobial susceptibility of the Acinetobacter calcoaceticus–Acinetobacter baumannii (Acb) species complex in Singapore. One hundred and ninety-three non-replicate Acb species complex clinical isolates were collected from six hospitals over a 1-month period in 2006. Of these, 152 (78·7%) were identified as A. baumannii, 18 (9·3%) as ‘Acinetobacter pittii’ [genomic species (gen. sp.) 3], and 23 (11·9%) as ‘Acinetobacter nosocomialis’ (gen. sp. 13TU). Carbapenem resistance was highest in A. baumannii (72·4%), followed by A. pittii (38·9%), and A. nosocomialis (34·8%). Most carbapenem-resistant A. baumannii and A. nosocomialis possessed the blaOXA-23-like gene whereas carbapenem-resistant A. pittii possessed the blaOXA-58-like gene. Two imipenem-resistant strains (A. baumannii and A. pittii) had the blaIMP-like gene. Representatives of carbapenem-resistant A. baumannii were related to European clones I and II.

scholarly journals Antibacterial effect of acetic acid during an outbreak of carbapenem-resistant Acinetobacter baumannii in an ICU (II)

2021 ◽  
Vol 15 (08) ◽  
pp. 1167-1172
Author(s):  
Carolina Garciglia-Mercado ◽  
Ramon Gaxiola-Robles ◽  
Felipe Ascencio ◽  
Concepción Grajales-Muñiz ◽  
Maria Luisa Soriano Rodríguez ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Acinetobacter Baumannii ◽  
Environmental Samples ◽  
Antibacterial Effect ◽  
Companion Paper ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Carbapenem Resistant ◽  
Before And After ◽  
Amplification Assay

Introduction: Acetic acid (AA) has been commonly used in medicine as an antiseptic agent for the past 6000 years. This study evaluated the antibacterial effect of AA during an outbreak in an intensive care unit (ICU) facility in Baja California Sur, México. Methodology: Thirty-five environmental samples were collected, subsequently, disinfection with AA (4%) was performed, and two days later the same areas were sampled inside the ICU facility. Carbapenem-resistant A. baumannii (CRAB) was detected with loop-mediated isothermal amplification assay (Garciglia-Mercado et al. companion paper), targeting blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP and blaVIM genes. CRAB isolates before and after disinfection were compared by PFGE. Results: Eighteen (54.5%) and five (14.3%) of thirty-five environmental samples were identified as Acinetobacter baumannii before and after disinfection, respectively, showing a significant decrease of 85.7% (p < 0.05) both by Loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Furthermore, the presence of blaOXA-23-like and blaOXA-58-like genes significantly decreased (p < 0.05) both by LAMP and PCR methods. PFGE genotype showed high similarity among CRAB isolates before and after disinfection, suggesting wide clonal dissemination in the ICU facility. Conclusions: This study demonstrated the novel application of AA with the LAMP assays developed for detecting CRAB. AA promises to be a cheap and efficacious disinfectant alternative to both developed and especially developing countries, preventing the spread of this organism in the environment and to other susceptible patients in health care settings.

Sign in / Sign up

Export Citation Format

Share Document