Circular presentation of the <i>mcr-1</i> containing <i>IncX4</i> plasmid in the colistin susceptible <i>E</i>.<i>coli</i>.

<p>In green the <i>mcr-1</i> sequence. In red the IS10R insertion sequence, interrupting the <i>mcr-1</i> gene at position 572. Arrows indicate open reading frames (ORFs), dark blue ORFs with annotation, light blue ORFs without annotation (hypothetical protein). Numbers indicate nucleotide positions.</p

Antibiotic phenotype with the corresponding molecular resistance of cultured <i>mcr-1</i> containing <i>E</i>.<i>coli</i>.

<p>Antibiotic phenotype with the corresponding molecular resistance of cultured <i>mcr-1</i> containing <i>E</i>.<i>coli</i>.</p

Primers and probe used to screen for the presence of <i>mcr</i>-genes.

<p>Primers and probe used to screen for the presence of <i>mcr</i>-genes.</p

Typing <i>Pseudomonas aeruginosa</i> Isolates with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry

The introduction of standardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms in the medical microbiological practice has revolutionized the way microbial species identification is performed on a daily basis. To a large extent, this is due to the ease of operation. Acquired spectra are compared to profiles obtained from cultured colonies present in a reference spectra database. It is fast and reliable, and costs are low compared to previous diagnostic approaches. However, the low resolution and dynamic range of the MALDI-TOF profiles have shown limited applicability for the discrimination of different bacterial strains, as achieved with typing based on genetic markers. This is pivotal in cases where certain strains are associated with, e.g., virulence or antibiotic resistance. Ultrahigh resolution MALDI-FTICR MS allows the measurement of small proteins at isotopic resolution and can be used to analyze complex mixtures with increased dynamic range and higher precision than MALDI-TOF MS, while still generating results in a similar time frame. Here, we propose to use ultrahigh resolution 15T MALDI-Fourier transform ion cyclotron resonance (FTICR) MS to discriminate clinically relevant bacterial strains after species identification performed by MALDI-TOF MS. We used a collection of well characterized <i>Pseudomonas aeruginosa</i> strains, featuring distinct antibiotic resistance profiles, and isolates obtained during hospital outbreaks. Following cluster analysis based on amplification fragment length polymorphism (AFLP), these strains were grouped into three different clusters. The same clusters were obtained using protein profiles generated by MALDI-FTICR MS. Subsequent intact protein analysis by electrospray ionization (ESI)-collision-induced dissociation (CID)-FTICR MS was applied to identify protein isoforms that contribute to the separation of the different clusters, illustrating the additional advantage of this analytical platform

Enhanced amplification of viral fragments using one restriction enzyme in VIDISCA.

<p>Visualization of VIDISCA fragments digested with <i>HinP1-I+MseI</i> or <i>MseI</i> alone. VIDISCA fragments are visualized on a 1% agarose gel, which were generated after a single first round PCR of 40 cycles. The dots indicate viral fragments which were only visible with <i>MseI</i> digestion.</p

Decrease of cDNA synthesis on rRNA templates.

a<p>In comparison to cDNA synthesis with all 4096 random hexamers.</p>b<p>In comparison to cDNA synthesis without rRNA-blocking oligo's.</p>c<p>In comparison to cDNA synthesis with all 4096 random hexamers and without rRNA-blocking oligo's.</p>d<p>binding region for blocking oligo 4-Morrna.</p>e<p>no rRNA-blocking oligo directed to this 1780–1880-region of 28S rRNA was added.</p>f<p>binding region for blocking oligo 3-Morrna.</p>g<p>binding region for blocking oligo 1-Morrna.</p

No decrease in viral genome amplification with random hexamers versus non-ribosomal hexamers.

<p>No decrease in viral genome amplification with random hexamers versus non-ribosomal hexamers.</p

Respiratory virus detection with VIDISCA-454.

<p>Respiratory virus detection with VIDISCA-454.</p

Enhanced viral RNA amplification in VIDISCA using non-ribosomal hexamers during reverse transcription.

<p>VIDISCA fragments are visualized on a 3% metaphor gel. A dilution series of echovirus 18 was used and the concentration per ml is indicated above each lane. NC = negative PBS control, M = 25 bp marker.(a) VIDISCA products were generated with primers Hinp-A/Mse-C. The viral fragments are 167 bp, 296 bp and 382 bp in size. (b) VIDISCA products amplified with primers Hinp-A/Mse-A. The product originating from rRNA (70 bp) is indicated by an arrow.</p

No inhibition of viral genome amplification with rRNA-blocking oligo's.

<p>No inhibition of viral genome amplification with rRNA-blocking oligo's.</p