scholarly journals SENSITIVITY OF KLEBSIELLA PNEUMONIAE CLINICAL ISOLATES WITH VARIOUS LEVELS OF ANTIBIOTIC RESISTANCE TO BACTERIORAGE PREPARATIONS

2018 ◽  
pp. 56-62
Author(s):  
D. V. Tapalsky ◽  
A. I. Kozlova
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
High Activity ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Isolates ◽  
Antibiotic Resistant ◽  
Medical Institutions ◽  
Different Levels

Objective : to assess sensitivity of K. pneumoniae clinical isolates with different levels of antibiotic resistance to commercial bacteriophage preparations. Material and methods . We have performed re-identification and determination of the sensitivity to antibiotics and bacteriophage preparations of 109 K. pneumoniae clinical isolates isolated from patients hospitalized in medical institutions of three regions of Belarus. The presence of carbapenemase genes has been deteсted by real-time PCR. Results . The study has shown awidespread prevalence of extreme antibiotic resistance among K. pneumoniae associated with the production of NDM and OXA-48 carbapenemases and has found an insufficient lytic activity of bacteriophage preparations against K. pneumoniae strains. The preparation «Sextafag», which lysed with a high activity of 28.4% of Klebsiella isolates possessed the widest spectrum of activity. Conclusion . Bacteriophage preparations can be considered as a possible alternative to antibiotics in the treatment of infections caused by extremely antibiotic-resistant K. pneumoniae isolates.It is necessary to supplement the composition of commercial preparations with phage strains that are active against Klebsiella isolates producing carbapenemases.

scholarly journals The impact of Grammosciadium platycarpum Boiss. & Hausskn. extract on oqxA efflux pump gene expression in antibiotic resistant clinical isolates of Klebsiella pneumoniae using real time PCR

2020 ◽  
Vol 19 (75) ◽  
pp. 291-304
Author(s):  
Faezeh Mohammadpour Bishak ◽  
Fatemeh Ashrafi ◽  
Soheila Moradi Bidhendi ◽  
Amir Mirzaie ◽  
Hassan Noorbazargan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Efflux Pump ◽  
Clinical Isolates ◽  
Antibiotic Resistant ◽  
The Impact

scholarly journals Use of Real-Time PCR and Fluorimetry for Rapid Detection of Rifampin and Isoniazid Resistance-Associated Mutations in Mycobacterium tuberculosis

2000 ◽  
Vol 38 (9) ◽  
pp. 3194-3199 ◽  
Author(s):  
Maria J. Torres ◽  
Antonio Criado ◽  
Jose C. Palomares ◽  
Javier Aznar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Antimicrobial Agents ◽  
Dna Amplification ◽  
Single Strand Conformation Polymorphism ◽  
Clinical Isolates ◽  
Polymorphism Analysis ◽  
Antibiotic Resistant ◽  
Hybridization Probes

Very fast amplification of DNA in small volumes can be continuously monitored with a rapid cycler that incorporates fluorimetric detection. Primers were designed to amplify a 157-bp fragment of therpoB gene spanning codons 526 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Mycobacterium tuberculosis. Most mutations associated with resistance to rifampin (RMP) and isoniazid (INH) in clinical isolates occur in these codons. Two pairs of hybridization probes were synthesized; one in each pair was 3′ labeled with fluorescein and hybridized upstream of the codon with the mutation; the other two probes were 5′ labeled with LightCycler-Red 640. Each pair of probes recognized adjacent sequences in the amplicon. After DNA amplification was finished by using a LightCycler, the temperature at which the Red 640 probe melted from the product was determined in a 3-min melt program. Twenty M. tuberculosis clinical isolates susceptible to streptomycin, INH, RMP, and ethambutol and 36 antibiotic-resistant clinical M. tuberculosis isolates (16 resistant to RMP, 16 to INH, and 4 to both antimicrobial agents) were amplified, and the presence of mutations was determined using single-strand conformation polymorphism analysis, the LiQor automated sequencer, and the LightCycler system. Concordant results were obtained in all cases. Within 30 min, the LightCycler method correctly genotyped all the strains without the need of any post-PCR sample manipulation. Overall, this pilot study demonstrated that real-time PCR coupled to fluorescence detection is the fastest available method for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.

A pentaplex real-time PCR assay for rapid identification of major beta-lactamase genes KPC, NDM, CTX, CMY, and OXA-48 directly from bacteria in blood

2021 ◽  
Vol 70 (12) ◽  
Author(s):  
Taalin R. Hoj ◽  
Bradley McNeely ◽  
Kylie Webber ◽  
Evelyn Welling ◽  
William G. Pitt ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Type Species ◽  
New Delhi ◽  
Beta Lactamase ◽  
Content Type ◽  
Link Type ◽  
Carbapenem Resistant

Introduction. Antibiotic resistance, particularly in cases of sepsis, has emerged as a growing global public health concern and economic burden. Current methods of blood culture and antimicrobial susceptibility testing of agents involved in sepsis can take as long as 3–5 days. It is vital to rapidly identify which antimicrobials can be used to effectively treat sepsis cases on an individual basis. Here, we present a pentaplex, real-time PCR-based assay that can quickly identify the most common beta-lactamase genes ( Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX-M); cephamycin AmpC beta-lactamases (CMY); and Oxacillinase-48 (OXA-48)) from pathogens derived directly from the blood of patients presenting with bacterial septicemia. Aim. To develop an assay which can rapidly identify the most common beta-lactamase genes in Carbapenem-resistant Enterobacteriaceae bacteria (CREs) from the United States. Hypothesis/Gap Statement. Septicemia caused by carbapenem-resistant bacteria has a death rate of 40–60 %. Rapid diagnosis of antibiotic susceptibility directly from bacteria in blood by identification of beta-lactamase genes will greatly improve survival rates. In this work, we develop an assay capable of concurrently identifying the five most common beta-lactamase and carbapenemase genes. Methodology. Primers and probes were created which can identify all subtypes of Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48). The assay was validated using 13 isolates containing various PCR targets from the Centre for Disease Control Antimicrobial Resistance Isolate Bank Enterobacterales Carbapenemase Diversity Panel. Blood obtained from volunteers was spiked with CREs and bacteria were separated, lysed, and subjected to analysis via the pentaplex assay. Results. This pentaplex assay successfully identified beta-lactamase genes derived from bacteria separated from blood at concentrations of 4–8 c.f.u. ml−1. Conclusion. This assay will improve patient outcomes by supplying physicians with critical drug resistance information within 2 h of septicemia onset, allowing them to prescribe effective antimicrobials corresponding to the resistance gene(s) present in the pathogen. In addition, information supplied by this assay will lessen the inappropriate use of broad-spectrum antimicrobials and prevent the evolution of further antibiotic resistance.

scholarly journals Real-Time PCR and Statistical Analyses of acrAB and ramA Expression in Clinical Isolates of Klebsiella pneumoniae

2008 ◽  
Vol 52 (9) ◽  
pp. 3430-3432 ◽  
Author(s):  
Alexey Ruzin ◽  
Frederick W. Immermann ◽  
Patricia A. Bradford
Keyword(s):  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Statistical Analyses ◽  
Clinical Isolates ◽  
Susceptibility Breakpoint

ABSTRACT Clinical isolates of Klebsiella pneumoniae were tested for a correlation between tigecycline MIC and expression of ramA by using real-time PCR. At MICs of 4 and 8 μg/ml, the expression of ramA was statistically significantly different from MICs of 2 μg/ml or less, supporting the tigecycline susceptibility breakpoint of ≤2 μg/ml for K. pneumoniae.

scholarly journals From the Farms to the Dining Table: The Distribution and Molecular Characteristics of Antibiotic-Resistant Enterococcus spp. in Intensive Pig Farming in South Africa

2021 ◽  
Vol 9 (5) ◽  
pp. 882
Author(s):  
Sasha Badul ◽  
Akebe L. K. Abia ◽  
Daniel G. Amoako ◽  
Keith Perrett ◽  
Linda A. Bester ◽  
...  
Keyword(s):  
South Africa ◽  
Antibiotic Resistance ◽  
Real Time ◽  
Real Time Pcr ◽  
Diffusion Method ◽  
Antibiotic Resistant ◽  
Pig Farming ◽  
Enterococcus Spp ◽  
Food Animals ◽  
The Continuum

Foodborne pathogens, including antibiotic-resistant species, constitute a severe menace to food safety globally, especially food animals. Identifying points of concern that need immediate mitigation measures to prevent these bacteria from reaching households requires a broad understanding of these pathogens’ spread along the food production chain. We investigated the distribution, antibiotic susceptibility, molecular characterization and clonality of Enterococcus spp. in an intensive pig production continuum in South Africa, using the farm-to-fork approach. Enterococcus spp. were isolated from 452 samples obtained along the pig farm-to-fork continuum (farm, transport, abattoir, and retail meat) using the IDEXX Enterolert®/Quanti-Tray® 2000 system. Pure colonies were obtained on selective media and confirmed by real-time PCR, targeting genus- and species-specific genes. The susceptibility to antibiotics was determined by the Kirby–Bauer disk diffusion method against 16 antibiotics recommended by the WHO-AGISAR using EUCAST guidelines. Selected antibiotic resistance and virulence genes were detected by real-time PCR. Clonal relatedness between isolates across the continuum was evaluated by REP-PCR. A total of 284 isolates, consisting of 79.2% E. faecalis, 6.7% E. faecium, 2.5% E. casseliflavus, 0.4% E. gallinarum, and 11.2% other Enterococcus spp., were collected along the farm-to-fork continuum. The isolates were most resistant to sulfamethoxazole-trimethoprim (78.8%) and least resistant to levofloxacin (5.6%). No resistance was observed to vancomycin, teicoplanin, tigecycline and linezolid. E. faecium displayed 44.4% resistance to quinupristin-dalfopristin. Also, 78% of the isolates were multidrug-resistant. Phenotypic resistance to tetracycline, aminoglycosides, and macrolides was corroborated by the presence of the tetM, aph(3′)-IIIa, and ermB genes in 99.1%, 96.1%, and 88.3% of the isolates, respectively. The most detected virulence gene was gelE. Clonality revealed that E. faecalis isolates belonged to diverse clones along the continuum with major REP-types, mainly isolates from the same sampling source but different sampling rounds (on the farm). E. faecium isolates revealed a less diverse profile. The results suggest that intensive pig farming could serve as a reservoir of antibiotic-resistant bacteria that could be transmitted to occupationally exposed workers via direct contact with animals or consumers through animal products/food. This highlights the need for more robust guidelines for antibiotic use in intensive farming practices and the necessity of including Enterococcus spp. as an indicator in antibiotic resistance surveillance systems in food animals.

scholarly journals Surveillance of potential pathogens and antibiotic resistance in wastewater and surface water from Boston, USA and Vellore, India using long-read metagenomic sequencing

2021 ◽  
Author(s):  
Erica R Fuhrmeister ◽  
Lee E Voth-Gaeddert ◽  
Angeline Metilda ◽  
Albert Tai ◽  
Rebecca E Batorsky ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Surface Water ◽  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Metagenomic Sequencing ◽  
Antibiotic Resistant ◽  
Short Read ◽  
Long Reads ◽  
Long Read

Environmental sampling (wastewater) could be an efficient surveillance strategy to capture global emerging trends in the spread of antibiotic resistance. Long-read DNA sequencing can resolve the genetic context of antibiotic resistance genes (ARGs) and is a promising tool for non- culture-based monitoring of antibiotic-resistant pathogens and ARGs in environmental samples, but has not been rigorously validated against conventional methods. We tested long-read sequencing using the portable Nanopore MinION for surveying pathogens, ARGs, and antibiotic- resistant pathogens in municipal wastewater, hospital wastewater, and surface water collected from Boston, USA and Vellore, India. We compared detection of enteric pathogens by assembly of long reads, with and without short-read polishing, and unassembled raw long reads for ARGs to multiplex real-time PCR. Using real-time PCR as a benchmark, long-read metagenomics was 49% sensitive and 75% specific at pathogen detection in assembled contigs, and 16% sensitive and 100% specific at detecting 28 clinically relevant resistance genes in raw long reads. Short- read polishing did not substantially improve pathogen identification or impact ARG identification in the assembled contigs, demonstrating that short-read polishing is not required, which greatly reduces costs. The high specificity of ARG detection supports portable long-read sequencing as a valuable tool to profile ARGs and antibiotic-resistant pathogens for environmental surveillance programs.

scholarly journals Network Integrative Genomic and Transcriptomic Analysis of Carbapenem-ResistantKlebsiella pneumoniaeStrains Identifies Genes for Antibiotic Resistance and Virulence

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Muyoung Lee ◽  
Naina Adren Pinto ◽  
Chan Yeong Kim ◽  
Sunmo Yang ◽  
Roshan D’Souza ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Carbapenem Resistance ◽  
Clinical Isolates ◽  
Functional Modules ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Functional Association ◽  
Carbapenem Resistant ◽  
Tract Infections

ABSTRACTGlobal increases in the use of carbapenems have resulted in several strains of Gram-negative bacteria acquiring carbapenem resistance, thereby limiting treatment options.Klebsiella pneumoniaeis a common carbapenem-resistant pathogenic bacterium that is widely studied to identify novel antibiotic resistance mechanisms and drug targets. Antibiotic-resistant clinical isolates generally harbor many genetic alterations, and the identification of responsible mutations would provide insights into the molecular mechanisms of antibiotic resistance. We propose a method to prioritize mutated genes responsible for antibiotic resistance on the basis of expression changes in their local subnetworks, hypothesizing that mutated genes that show significant expression changes among the corresponding functionally associated genes are more likely to be involved in the carbapenem resistance. For network-based gene prioritization, we developed KlebNet (www.inetbio.org/klebnet), a genome-scale cofunctional network ofK. pneumoniaegenes. Using KlebNet, we reconstructed the functional modules for carbapenem resistance and virulence and identified the functional association between antibiotic resistance and virulence. Using complementation assays with the top candidate genes, we were able to validate a novel gene that negatively regulated carbapenem resistance and four novel genes that positively regulated virulence inGalleria mellonellalarvae. Therefore, our study demonstrated the feasibility of network-based identification of genes required for antibiotic resistance and virulence of human-pathogenic bacteria.IMPORTANCEKlebsiella pneumoniaeis a major bacterial pathogen that causes pneumonia and urinary tract infections in human.K. pneumoniaeinfections are treated with carbapenem, but carbapenem-resistantK. pneumoniaehas been spreading worldwide. We are able to identify antimicrobial-resistant genes among mutated genes of the antibiotic-resistant clinical isolates. However, they usually harbor many mutated genes, including those that cause weak or neutral functional effects. Therefore, we need to prioritize the mutated genes to identify the more likely candidates for the follow-up functional analysis. For this study, we present a functional network ofK. pneumoniaegenes and propose a network-based method of prioritizing the mutated genes of the resistant clinical isolates. We also reconstructed the network-based functional modules for carbapenem resistance and virulence and retrieved the functional association between antibiotic resistance and virulence. This study demonstrated the feasibility of network-based analysis of clinical genomics data for the study ofK. pneumoniaeinfection.

scholarly journals Multiplex Real-Time PCR for Detection of an Epidemic KPC-Producing Klebsiella pneumoniae ST258 Clone

2012 ◽  
Vol 56 (6) ◽  
pp. 3444-3447 ◽  
Author(s):  
Liang Chen ◽  
Kalyan D. Chavda ◽  
José R. Mediavilla ◽  
Yanan Zhao ◽  
Henry S. Fraimow ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Isolates ◽  
Pcr Assay ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Sequence Types ◽  
Single Reaction

ABSTRACTWe describe a multiplex real-time PCR assay capable of identifying both the epidemicKlebsiella pneumoniaeST258 clone andblaKPCcarbapenemase genes in a single reaction. The assay displayed excellent sensitivity (100%) and specificity (100%) for identification of ST258 clone andblaKPCin a collection of 75K. pneumoniaeisolates comprising 41 sequence types. Our results suggest that this assay is an effective tool for surveillance of this clone among carbapenem-resistantK. pneumoniaeclinical isolates.

scholarly journals Antibiotic Resistance Among Klebsiella pneumoniae, Molecular Detection and Expression Level of blaKPC and blaGES Genes by Real-Time PCR

2019 ◽  
Vol 12 (10) ◽  
Author(s):  
Narjes Mohammadi Bandari ◽  
Mohsen Zargar ◽  
Hossein Keyvani ◽  
Malihe Talebi ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Molecular Detection ◽  
Expression Level
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