Novel multiplex PCRs for detection of the most prevalent carbapenemase genes in Gram-negative bacteria within Germany

2021 ◽  
Author(s):  
Mónica Cerezales ◽  
Lea Biniossek ◽  
Stefanie Gerson ◽  
Kyriaki Xanthopoulou ◽  
Julia Wille ◽  
...  
Keyword(s):  
Carbapenem Resistance ◽  
Clinical Isolates ◽  
Common Source ◽  
Reference Laboratory ◽  
Gram Negative Bacteria ◽  
Simple Method ◽  
Gram Negative ◽  
Multiplex Pcrs ◽  
Carbapenem Resistant ◽  
Encoding Genes

Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants. Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available. Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany. Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM, bla OXA-40, bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51, bla IMI, bla FIM and bla DIM. We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM and bla OXA-40, while multiplex-2 included bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51 and bla IMI. Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected. Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.

scholarly journals An overview of carbapenem, its resistance and therapeutic options for infections caused by carbapenem resistant bacteria

2020 ◽  
Vol 9 (9) ◽  
pp. 1454
Author(s):  
Hari P. Nepal ◽  
Rama Paudel
Keyword(s):  
Bacterial Infections ◽  
Carbapenem Resistance ◽  
Therapeutic Options ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Beta Lactam ◽  
Gram Negative ◽  
Penicillin Binding Proteins ◽  
Carbapenem Resistant ◽  
The World

Carbapenems are beta-lactam drugs that have broadest spectrum of activity. They are commonly used as the drugs of last resort to treat complicated bacterial infections. They bind to penicillin binding proteins (PBPs) and inhibit cell wall synthesis in bacteria. Important members that are in clinical use include doripenem, ertapenem, imipenem, and meropenem. Unlike other members, imipenem is hydrolyzed significantly by renal dehydropeptidase; therefore, it is administered together with an inhibitor of renal dehydropeptidase, cilastatin. Carbapenems are usually administered intravenously due to their low oral bioavailability. Most common side effects of these drugs include nausea, vomiting, diarrhea, skin rashes, and reactions at the infusion sites. Increasing resistance to these antibiotics is being reported throughout the world and is posing a threat to public health.  Primary mechanisms of carbapenem resistance include expulsion of drug and inactivation of the drug by production of carbapenemases which may not only hydrolyze carbapenem, but also cephalosporin, penicillin, and aztreonam. Resistance especially among Gram negative bacteria is of much concern since there are only limited therapeutic options available for infections caused by carbapenem resistant Gram-negative bacterial pathogens. Commonly used drugs to treat such infections include polymyxins, fosfomycin and tigecycline.

scholarly journals Genome-Wide Identification of Carbapenem-Resistant Gram- Negative Bacterial (CR-GNB) Isolates Retrieved From Hospitalized Patients in Bihar, India

2021 ◽  
Author(s):  
Namrata Kumari ◽  
Mukesh Kumar ◽  
Amit Katiyar ◽  
Abhay Kumar ◽  
Pallavi Priya ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Carbapenem Resistance ◽  
Cross Sectional Study ◽  
Clinical Isolates ◽  
Cross Sectional ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Genome Wide ◽  
Synergy Test ◽  
Global Threat

Abstract Carbapenemase-producing clinical isolates are becoming more common over the world, posing a severe public health danger, particularly in developing nations like India. Carbapenem-resistant Gram-negative bacterial (CR-GNB) infection has become a fast-expending global threat with limited antibiotic choice and significant mortality. The aim of this study was to highlight the carbapenem-resistance among clinical isolates of hospital admitted patients in Bihar, India. A cross-sectional study was conducted with 101 clinical isolates of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa. All GNB isolates were tested for their antimicrobial susceptibility using double disc synergy test / modified hodge test (DDST/MHT) and subsequently confirmed carbapenemase-producing isolates were evaluated for carbapenem-resistance genes using whole-genome sequencing (genotypically) method. The overall percentage of carbapenem-resistance among GNB was (17/101) 16.83%. The AMR analysis demonstrates a significantly high prevalence of blaCTX−M followed by blaSHV, blaTEM, blaOXA and blaNDM β-lactams carbapenem-resistance genes among clinical isolates of GNB. Co-occurrence of carbapenemase-encoding genes with blaNDM was found in 70.6% of carbapenemase-producing isolates. Our study highlights the mechanism of carbapenem-resistance to curb the overwhelming threat posed by emergence of drug-resistance in India.

Determination of carbapenemase genes in clinical isolates using Cepheid Xpert Carba-R

2021 ◽  
pp. 16-19
Author(s):  
N. I. Gabrielyan ◽  
V. G. Kormilitsyna ◽  
V. K. Zaletaeva ◽  
A. V. Krotevich ◽  
I. A. Miloserdov ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Infection Control ◽  
Resistance Genes ◽  
Carbapenem Resistance ◽  
Critical Issue ◽  
Sensitivity Study ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Carbapenem Resistant

Detection of carbapenem resistance genes is a critical issue for hospitals due to possible recommendations for infection control and targeted therapy. The Cepheid Xpert instrument, a Carba-R test for the detection and differentiation of five common carbapenemase genes, was tested from September 2020 to February 2021. As part of the approbation, 20 tests were provided. This review presents the results of the approbation of a relatively regular sensitivity study on Siemens WalkAway‑96 plus. Cepheid Xpert Carba-R analysis has been shown to be an accurate and fast tool for detecting colonization by carbapenem-resistant gram-negative bacteria, which can help limit the spread of these organisms in hospitals.

scholarly journals Detection and Characterization of Carbapenem resistant Gram-negative bacilli isolates recovered from hospitalized patients at Soba University Hospital, Sudan

2020 ◽  
Author(s):  
Hana S. Elbadawi ◽  
Kamal M. Elhag ◽  
Elsheikh Mahgoub ◽  
Hisham N Altayb ◽  
Francine Ntoumi ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Nosocomial Infections ◽  
Carbapenem Resistance ◽  
Clinical Isolates ◽  
University Hospital ◽  
P Value ◽  
Relative Distribution ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Carbapenemase Gene

Abstract Background:Antimicrobial resistance (AMR) poses a threat to global health security. Whilst over the past decade, there has been an increase in reports of nosocomial infections globally caused by carbapenem resistant Gram-negative bacilli (GNB), data from Africa have been scanty. We performed a study of carbapenem resistance genes among GNB isolated from patients treated in hospitals in Khartoum state, Sudan.Methods:A cross-sectional study was conducted at Soba University Hospital (SUH) and Institute of Endemic Diseases, University of Khartoum for the period October 2016 to February 2017. A total of 206 GNB isolates from different clinical specimens were analyzed for carbapenem resistance genes using phenotypic tests and affirmed by genes detection. Multiplex PCR was performed for each strain to detect the carbapenemase genes, including the blaNDM, blaVIM, blaIMP, blaKPC, and blaOXA-48. In addition to blaCTXM, blaTEM and blaSHV. DNA sequencing and bioinformatics analysis were used to detect genes subtypes.Findings:Of 206 isolates, 171 (83%) were confirmed resistant phenotypically and 121 (58.7%) isolates were positive for the presence of one or more carbapenemase gene. New Delhi metallo-β-lactamase (NDM) types were the most predominant genes, blaNDM 107(88.4%). Others included blaIMP 7 (5.7%), blaOXA-48 5(4.1%), blaVIM 2 (1.6%) and blaKPC 0 (0%). Co- resistance genes with NDM producing GNB were detected in 87 (81.3%) of all blaNDM positive isolates. A significant association between phenotypic and genotypic resistance was observed (P- value < 0.001). NDM-1 was the most sub type was observed in 75 isolates (70 %), other subtypes were NDM- 5 and NDM-6. Infections due to Carbapenem resistant GNB are increasing at SUH, with the blaNDM being the prevalent genes among clinical isolates and belong to the Indian lineage.Conclusions:The frequency of carbapenemase producing bacilli was found to be improperly high in Khartoum hospitals. NDM was found to be the most prevalent carbapenemase gene among clinical isolates. Close surveillance across all hospitals in Sudan is required. The relative distribution of Carbapenemase genes among GNB in nosocomial infections in Africa needs to be defined.

scholarly journals Performance of the BD Phoenix CPO detect assay for detection and classification of carbapenemase-producing organisms

2020 ◽  
Author(s):  
Laura Berneking ◽  
Anna Both ◽  
Benjamin Berinson ◽  
Armin Hoffmann ◽  
Marc Lütgehetmann ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Carbapenem Resistance ◽  
Alternative Methods ◽  
Gram Negative Bacteria ◽  
Accurate Identification ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Gradient Diffusion ◽  
Assay Performance

AbstractIncreasing worldwide, prevalence of carbapenem-resistant gram-negative bacteria demands urgent a need for rapid detection and accurate identification of carbapenemases. The BD Phoenix CPO detect (PCD) assay possesses an in-built capacity for parallel susceptibility testing and detection of carbapenemases. Here, the ability of the assay to detect and classify carbapenemase production was tested in a collection of carbapenem-resistant Enterobacterales and non-fermentative gram-negative rods. The ability of the PCD assay to detect and classify carbapenemases was investigated in a collection of 194 clinical, carbapenem-resistant isolates (Enterobacterales [n = 65]; non-fermentative gram-negative rods [n = 129]). AST results were compared to MICS determined by gradient diffusion to determine accuracy of the PCD assay. The accuracy of the PCD assay to detect carbapenemases was compared to the results of molecular isolate characterization using a LDT multiplex carbapenemase PCR assay. All 194 isolates classified as carbapenem-resistant by reference susceptibility testing were also classified correctly as CRO by the PCD assay. Performance analysis of the PCD assay to detect carbapenemase production revealed an overall sensitivity of 98.29% and specificity of 17.95% for the detection of carbapenemase production. For the classification of carbapenemases classes A, B, and D, the PCD correctly classified 79.17% Enterobacterales and 67.16% non-fermentative gram-negative rods. The PCD assay is a reliable tool for the detection of carbapenem resistance and allows for parallel analysis of carbapenemase production. However, while sensitivity is high, low specificity in carbapenemase detection and erroneous classification demands mandatory confirmation by alternative methods, especially in non-fermentative gram-negative bacteria.

scholarly journals Identification of blaGIM-1 in Acinetobacter variabilis isolated from the hospital environment in Tamil Nadu, India

2019 ◽  
Author(s):  
Prasanth Manohar ◽  
Murugavel Ragavi ◽  
Ashby Augustine ◽  
Hrishikesh MV ◽  
Nachimuthu Ramesh
Keyword(s):  
Tamil Nadu ◽  
Carbapenem Resistance ◽  
Resistance Mechanisms ◽  
Hospital Environment ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Hospital Environments ◽  
Surveillance Programs

AbstractBackgroundEmergence of carbapenem resistance mechanisms among Gram-negative bacteria is a worrisome health problem. Here, we focused on to identify the presence of carbapenem-resistant bacteria among the samples collected from hospital environments in Tamil Nadu.MethodsA total of 30 hospital environmental samples were collected between August 2017 and January 2018 from hospitals located in Chennai and Vellore such as lift switches, stair rails, switchboards, nursing desks, used nursing gloves, door handles, wheelchairs, touch screens, chairs and from pillars inside the hospitals.Results and discussionA total of 22 carbapenem-resistant Gram-negative bacteria were isolated that included Escherichia coli, Klebsiella sp., Enterobacter sp., Salmonella sp., Pseudomonas aeruginosa and Acinetobacter sp. Interestingly, blaGIM-1 was detected in Acinetobacter variabilis strain isolated in samples collected from hospitals. Unlike other studies, the identified GIM-1 was not plasmid encoded, and this is the first report for the presence of GIM-1 (German imipenemase) in India.ConclusionExtensive surveillance programs are necessary to trace the uncontrolled spread of carbapenem-resistance genes in order to reduce the rapid spread of resistance.

scholarly journals Direct Testing for KPC-Mediated Carbapenem Resistance from Blood Samples Using a T2 Magnetic Resonance Based Assay

Antibiotics ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 950
Author(s):  
Giulia De Angelis ◽  
Riccardo Paggi ◽  
Thomas J. Lowery ◽  
Jessica L. Snyder ◽  
Giulia Menchinelli ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Carbapenem Resistance ◽  
Bloodstream Infections ◽  
Resistance Testing ◽  
Blood Samples ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Direct Testing ◽  
Two Samples ◽  
Encoding Genes

Molecular-based carbapenem resistance testing in Gram-negative bacterial bloodstream infections (BSIs) is currently limited because of the reliance on positive blood culture (BC) samples. The T2Resistance™ panel may now allow the detection of carbapenemase- and other β-lactamase encoding genes directly from blood samples. We detected carbapenem resistance genes in 11 (84.6%) of 13 samples from patients with BC-documented BSIs (10 caused by KPC-producing Klebsiellapneumoniae and 1 caused by VIM/CMY-producing Citrobacter freundii). Two samples that tested negative for carbapenem resistance genes were from patients with BC-documented BSIs caused by KPC-producing K. pneumoniae who were receiving effective antibiotic therapy. In conclusion, our findings suggest that the T2Resistance™ panel can be a reliable tool for diagnosing carbapenem-resistant Gram-negative bacterial BSIs.

scholarly journals Carbapenem Resistance in Gram-negative Bacteria in South-western Nigeria: The Role of Extended-spectrum β-lactamase CTX-M-15

2018 ◽  
pp. 344-349
Author(s):  
Do Ogbolu ◽  
Ma Webber
Keyword(s):  
Outer Membrane ◽  
Antimicrobial Agents ◽  
Random Amplified Polymorphic Dna ◽  
Outer Membrane Proteins ◽  
Carbapenem Resistance ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Extended Spectrum

Objective: To determine the role of extended-spectrum β-lactamases in carbapenem-resistant Gram-negative bacteria from south-western Nigeria. Methods: Twenty-seven carbapenem-resistant isolates that were found to be non-carbapenemase producers (15 Escherichia coli, 9 Klebsiella pneumoniae and 3 Pseudomonas aeruginosa) were further studied. These isolates were subjected to analysis including phenotypic and genotypic detection of various β-lactamases, efflux activity, outer membrane protein, plasmids replicon typing, detection of transferable genes and resistances and typing using random amplified polymorphic DNA tests. Results: No isolates demonstrated de-repression of efflux, but all showed either complete loss or reduced production of outer membrane proteins. Transconjugants from these strains contained various genes including plasmid-mediated quinolone resistance and extended-spectrum beta-lactamases. All the transconjugants carried the blaCTX-M-15 gene. The transconjugants had varying minimum inhibitory concentrations of carbapenems ranging from 0.03 μg/ml to 8 μg/ml. Varying resistances to other antimicrobial agents were also transferred with the plasmids. The donor isolates were not clonally related by molecular typing. Conclusion: Resistance to carbapenem antibiotics in this sample was not mediated only by carbapenemases but also by production of extended-spectrum β-lactamases (largely CTX-M-15), accompanied by protein loss. This was an important mechanism underpinning carbapenem resistance in these clinical isolates of various species.

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