Rapid Identification of Carbapenemase Genes in Gram-Negative Bacteria with an Oligonucleotide Microarray-Based Assay

<div><p>Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, <i>Citrobacter freundii/braakii</i>, <i>Klebsiella pneumoniae</i> and <i>Acinetobacter baumannii</i>. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included <i>bla</i>VIM (13 out of 13), <i>bla</i>GIM (2/2), <i>bla</i>KPC (27/27), <i>bla</i>NDM (5/5), <i>bla</i>IMP-2/4/7/8/13/14/15/16/31 (10/10), <i>bla</i>OXA-23 (12/13), <i>bla</i>OXA-40-group (7/7), <i>bla</i>OXA-48-group (32/33), <i>bla</i>OXA-51 (1/1) and <i>bla</i>OXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [<i>bla</i>OXA-1 (16/16), <i>bla</i>OXA-2 (4/4), <i>bla</i>OXA-9 (33/33), OXA-10 (3/3), <i>bla</i>OXA-51 (25/25), <i>bla</i>OXA-58 (2/2), CTX-M1/M15 (17/17) and <i>bla</i>VIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including <i>Acinetobacter baumannii</i> (28/28), <i>Enterobacter spec</i>. (5/5), <i>Escherichia coli</i> (4/4), <i>Klebsiella pneumoniae</i> (62/63), <i>Klebsiella oxytoca</i> (0/2), <i>Pseudomonas aeruginosa</i> (12/12), <i>Citrobacter freundii</i> (1/1) and <i>Citrobacter braakii</i> (2/2), were correctly identified by a panel of species specific probes. This assay might be easily extended, adapted and transferred to point of care platforms enabling fast surveillance, rapid detection and appropriate early treatment of infections caused by multiresistant Gram-negative bacteria.</p></div

Linear multiplex DNA amplification, labeling and hybridization with the ArrayStrips.

<p>(<b>a</b>) Linear Multiplex Amplification starting from clonal RNA-free genomic DNA. Extracted DNA is internally labeled with biotin (Label [L]) and amplified in a linear multiplex PCR reaction; (<b>b</b>) Hybridization: the internally biotin labeled, single-stranded DNA product hybridizes specifically under stringent conditions to the corresponding probes. The resulting duplex is detected using a horse-radish peroxidase (Enzyme [E]) – streptavidin conjugate, which causes the dye to precipitate ([S]). (<b>c</b>) Detection: the ArrayMate Reader (or ArrayTube Reader ATR 03) enables the visualization and subsequently automated analysis of the array image. The presence of a dark precipitated spot indicates successful hybridization; (<b>d</b>) Analysis: the assay specific software analysis script coming with the ArrayMate Reader (or ArrayTube Reader ATR 03), measures the signal intensity of each probe and determines which genes/alleles are present in the sample by means of an assay specific algorithm. (<b>e</b>) Genotype analysis: a software plugin coming with the ArrayMate Reader (or ArrayTube Reader ATR 03) analyzed the raw data automatically, finally a report is provided on the detected carbapenemases genes.</p

Additional lactamases detected by the microarray in comparison to conducted control PCRs.

<p>Additional lactamases detected by the microarray in comparison to conducted control PCRs.</p

Species genotyping results of the <b>M</b>icroarray-based assay in comparison to the <b>R</b>eference method (phenotypic results were obtained from University Medical Center of Dresden, University Medical Center of Jena, German Collection of Microorganisms and Cell Cultures, Institut Pasteur and Friedrich-Loeffler-Institute).

<p>Species genotyping results of the <b><u>M</u></b>icroarray-based assay in comparison to the <b><u>R</u></b>eference method (phenotypic results were obtained from University Medical Center of Dresden, University Medical Center of Jena, German Collection of Microorganisms and Cell Cultures, Institut Pasteur and Friedrich-Loeffler-Institute).</p

Target overview of the microarray-based carbapenemases assay.

<p>Target overview of the microarray-based carbapenemases assay.</p

Carbapenemases genotyping results of the <b>M</b>icroarray-based assay in comparison to the genotyping results of carbapenemases genes obtained from different reference laboratories that used standard PCR as <b>R</b>eference method.

<p>Carbapenemases genotyping results of the <b><u>M</u></b>icroarray-based assay in comparison to the genotyping results of carbapenemases genes obtained from different reference laboratories that used standard PCR as <b><u>R</u></b>eference method.</p

Table_1_Molecular Typing of ST239-MRSA-III From Diverse Geographic Locations and the Evolution of the SCCmec III Element During Its Intercontinental Spread.PDF

<p>ST239-MRSA-III is probably the oldest truly pandemic MRSA strain, circulating in many countries since the 1970s. It is still frequently isolated in some parts of the world although it has been replaced by other MRSA strains in, e.g., most of Europe. Previous genotyping work (Harris et al., 2010; Castillo-Ramírez et al., 2012) suggested a split in geographically defined clades. In the present study, a collection of 184 ST239-MRSA-III isolates, mainly from countries not covered by the previous studies were characterized using two DNA microarrays (i) targeting an extensive range of typing markers, virulence and resistance genes and (ii) a SCCmec subtyping array. Thirty additional isolates underwent whole-genome sequencing (WGS) and, together with published WGS data for 215 ST239-MRSA-III isolates, were analyzed using in-silico analysis for comparison with the microarray data and with special regard to variation within SCCmec elements. This permitted the assignment of isolates and sequences to 39 different SCCmec III subtypes, and to three major and several minor clades. One clade, characterized by the integration of a transposon into nsaB and by the loss of fnbB and splE was detected among isolates from Turkey, Romania and other Eastern European countries, Russia, Pakistan, and (mainly Northern) China. Another clade, harboring sasX/sesI is widespread in South-East Asia including China/Hong Kong, and surprisingly also in Trinidad & Tobago. A third, related, but sasX/sesI-negative clade occurs not only in Latin America but also in Russia and in the Middle East from where it apparently originated and from where it also was transferred to Ireland. Minor clades exist or existed in Western Europe and Greece, in Portugal, in Australia and New Zealand as well as in the Middle East. Isolates from countries where this strain is not epidemic (such as Germany) frequently are associated with foreign travel and/or hospitalization abroad. The wide dissemination of this strain and the fact that it was able to cause a hospital-borne pandemic that lasted nearly 50 years emphasizes the need for stringent infection prevention and control and admission screening.</p