Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

ABSTRACT We directly tested 484 organisms from clinical (n = 310) and simulated (n = 174) positive blood cultures using the NG-Test Carba 5 assay for carbapenemase-producing Enterobacterales detection. The assay identified all but 4 of the KPC (170/171), OXA-48-like (22/22), VIM (19/21), and NDM (14/15) producers with no false positives. Among the clinical Klebsiella pneumoniae organisms tested, 122 of 123 KPC, 1 of 1 OXA-48-like, and 1 of 2 VIM producers were detected by the assay. Some VIM and NDM producers yielded scant but still-readable bands with the assay. No organisms produced the IMPs that the assay was designed to detect.

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Evaluation of differential gene expression in susceptible and resistant clinical isolates of Klebsiella pneumoniae by DNA microarray analysis

Clinical Microbiology and Infection ◽

10.1111/j.1469-0691..01470.x ◽

2006 ◽

Vol 12 (9) ◽

pp. 936-940 ◽

Cited By ~ 10

Author(s):

A. Doménech-Sánchez ◽

V. Javier Benedí ◽

L. Martínez-Martínez ◽

S. Albertí

Keyword(s):

Gene Expression ◽

Klebsiella Pneumoniae ◽

Differential Gene Expression ◽

Microarray Analysis ◽

Dna Microarray ◽

Clinical Isolates ◽

Dna Microarray Analysis ◽

Differential Gene

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Occurrence of the p019 gene in the blaKPC-harbouring plasmids: adverse clinical impact for direct tracking of KPC-producing Klebsiella pneumoniae by MALDI-TOF MS

Journal of Clinical Microbiology ◽

10.1128/jcm.00238-21 ◽

2021 ◽

Author(s):

Eva Gato ◽

Ignacio Pedro Constanso ◽

Bruno Kotska Rodiño-Janeiro ◽

Paula Guijarro-Sánchez ◽

Tyler Alioto ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Computational Analysis ◽

Direct Detection ◽

Clinical Impact ◽

Mass Peak ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Direct Tracking ◽

Geographical Locations ◽

Tof Ms

MALDI-TOF MS has recently been used for the direct detection of KPC-producing isolates by analysis of the 11,109 Da mass peak representing the P019 protein. In this study we evaluate the presence of the 11,109 Da mass peak in a collection of 435 unduplicated K. pneumoniae clinical isolates. The prevalence of the P019 peak in the blaKPC K. pneumoniae isolates was 49.2% (32/65). The 11,109 Da mass peak was not observed in any of the other carbapenemase (319) or non carbapenemase producers (116). Computational analysis of the presence of the p019 gene was performed in the aforementioned carbapenemase-producing K. pneumoniae isolates fully characterized by WGS and in a further collection of 1,649 K. pneumoniae genomes included in EuSCAPE. Herein, we have demonstrated that the p019 gene is not exclusively linked to the pKpQil plasmid, but it is present in the following plasmids: IncFIB(K)/IncFII(K)/ColRNAI, IncFIB(pQil), IncFIB(pQil)/ColRNAI, IncFIB(pQil)/IncFII(K), IncFIB(K)/IncFII(K) and IncX3. Besides, we have proven the independent movement of the Tn4401 and the ISKpn31, of which the p019 gene is a component. The absence of the p019 gene was obvious in Col440I, Col(pHAD28), IncFIB(K)/IncX3/IncFII(K), IncFIB(K)/IncFII(K) plasmids. In addition, we also observed another plasmid in which neither Tn4401 nor ISKpn31 was found, IncP6. In the EuSCAPE, the occurrence of p019 varied from 0% to 100% among the different geographical locations. The adverse clinical impact of the diminished prevalence of the p019 gene within the plasmid encoding KPC-producing Klebsiella pneumoniae puts forward the need for reconsideration when applying this technique in a clinical setting.

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Carbapenemase Producing Klebsiella pneumoniae (KPC): What Is the Best MALDI-TOF MS Detection Method

Antibiotics ◽

10.3390/antibiotics10121549 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1549

Author(s):

Lukáš Hleba ◽

Miroslava Hlebová ◽

Anton Kováčik ◽

Juraj Čuboň ◽

Juraj Medo

Keyword(s):

Mass Spectrum ◽

Klebsiella Pneumoniae ◽

Direct Detection ◽

Degradation Products ◽

Hospital Setting ◽

Morbidity And Mortality ◽

Klebsiella Pneumoniae Carbapenemase ◽

The Third ◽

Maldi Tof ◽

Positive Mode

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria is a group of highly dangerous antibiotic resistant Gram-negative Enterobacteriaceae. They cause infections associated with significant morbidity and mortality. Therefore, the rapid detection of KPC-producing bacteria plays a key role in clinical microbiology. Matrix assisted laser desorption/ionization time-of- flight (MALDI-TOF) is a rapidly evolving technology that finds application in various clinical, scientific, and industrial disciplines. In the present study, we demonstrated three different procedures of carbapenemase-producing K. pneumoniae (KPC) detection. The most basic model of MALDI-TOF instrument MS Microflex LT was used, operating in the linear ion-positive mode, commonly used in modern clinical laboratories. The first procedure was based on indirect monitoring of carbapenemase production with direct detection of hydrolyzed carbapenem antibiotic degradation products in the mass spectrum. The second procedure was based on direct detection of blaKPC accompanying peak with an 11,109 Da in the mass spectrum of carbapenemase-producing K. pneumoniae (KPC), which represents the cleaved protein (pKpQIL_p019) expressed by pKpQIL plasmid. In addition, several unique peaks were detected in the carbapenemase-producing K. pneumoniae (KPC) mass spectrum. The third procedure was the identification of carbapenemase-producing K. pneumoniae (KPC) based on the protein fingerprint using local database created from the whole mass spectra. By comparing detection procedures, we determined that the third procedure was very fast and relatively easy. However, it requires previous verification of carbapenemase-producing K. pneumoniae (KPC) using other methods as genetic blaKPC identification, detection of carbapenem degradation products, and accompanying peak with 11,109 Da, which represents cleaved pKpQIL_p019 protein expressed by pKpQIL plasmid. Detection of carbapenemase-producing K. pneumoniae using MALDI-TOF provides fast and accurate results that may help to reduce morbidity and mortality in hospital setting when applied in diagnostic situations.

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A DNA Microarray Platform Based on Direct Detection of rRNA for Characterization of Freshwater Sediment-Related Prokaryotic Communities

Applied and Environmental Microbiology ◽

10.1128/aem.02949-05 ◽

2006 ◽

Vol 72 (7) ◽

pp. 4829-4838 ◽

Cited By ~ 31

Author(s):

Jörg Peplies ◽

Christine Lachmund ◽

Frank Oliver Glöckner ◽

Werner Manz

Keyword(s):

16S Rrna ◽

Dna Microarray ◽

Bacterial Communities ◽

Direct Detection ◽

Signal To Noise Ratio ◽

Microarray Platform ◽

Freshwater Sediments ◽

Pure Cultures ◽

Catalyzed Reporter Deposition

ABSTRACT A DNA microarray platform for the characterization of bacterial communities in freshwater sediments based on a heterogeneous set of 70 16S rRNA-targeted oligonucleotide probes and directly labeled environmental RNA was developed and evaluated. Application of a simple protocol for the efficient background blocking of aminosilane-coated slides resulted in an improved signal-to-noise ratio and a detection limit of 10 ng for particular 16S rRNA targets. An initial specificity test of the system using RNA from pure cultures of different phylogenetic lineages showed a fraction of false-positive signals of ∼5% after protocol optimization and a marginal loss of correct positive signals. Subsequent microarray analysis of sediment-related community RNA from four different German river sites suggested low diversity for the groups targeted but indicated distinct differences in community composition. The results were supported by parallel fluorescence in situ hybridization in combination with sensitive catalyzed reporter deposition (CARD-FISH). In comparisons of the data of different sampling sites, specific detection of populations with relative cellular abundances down to 2% as well as a correlation of microarray signal intensities and population size is suggested. Our results demonstrate that DNA microarray technology allows for the fast and efficient precharacterization of complex bacterial communities by the use of standard single-cell hybridization probes and the direct detection of environmental rRNA, also in methodological challenging habitats such as heterogeneous lotic freshwater sediments.

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