Rapid and Simultaneous Detection of \u3ci\u3ebla\u3c/i\u3e\u3csub\u3eKPC\u3c/sub\u3e and \u3ci\u3ebla\u3c/i\u3e\u3csub\u3eNDM\u3c/sub\u3e by Use of Multiplex Real-Time PCR

The increasing incidence of carbapenem nonsusceptibility among clinically important species is of global concern. Identification of the molecular mechanisms underlying carbapenem nonsusceptibility is critical for epidemiological investigations. In this report, we describe a real-time PCR-based assay capable of simultaneously detecting blaKPC and blaNDM, two of the most important carbapenemases, directly from culture in less than 90 min. The assay was validated with blaKPC- and blaNDM-carrying clinical isolates and demonstrated 100% concordance with the Carba NP test

Novel 16S rRNA Methyltransferase RmtH Produced by \u3ci\u3eKlebsiella pneumoniae\u3c/i\u3e Associated with War-Related Trauma

Klebsiella pneumoniae strain MRSN2404 was isolated from the chronic wound of a soldier who had been wounded in Iraq in 2006. The strain displayed very high MICs of all aminoglycosides, including arbekacin. A gene encoding a novel 16S rRNA methyltransferase, now designated RmtH, was identified. RmtH had 64% identity with RmtB1 and RmtB2. rmtH was bracketed by two copies of ISCR2, which may have played a role in its mobilization

Bacterial Peritonitis Due to \u3ci\u3eAcinetobacter baumannii\u3c/i\u3e Sequence Type 25 with Plasmid-Borne New Delhi Metallo-Beta-Lactamase in Honduras

A carbapenem-resistant Acinetobacter baumannii strain was isolated from the peritoneal fluid of a patient with complicated intra-abdominal infection and evaluated at the Multidrug-resistant Organism Repository and Surveillance Network by wholegenome sequencing and real-time PCR. The isolate was sequence type 25 and susceptible to colistin and minocycline, with low MICs of tigecycline. blaNDM-1 was located on a plasmid with \u3e99% homology to pNDM-BJ02. The isolate carried numerous other antibiotic resistance genes, including the 16S methylase gene, armA

Enhanced De Novo Assembly of High Throughput Pyrosequencing Data Using Whole Genome Mapping

<div><p>Despite major advances in next-generation sequencing, assembly of sequencing data, especially data from novel microorganisms or re-emerging pathogens, remains constrained by the lack of suitable reference sequences. <i>De novo</i> assembly is the best approach to achieve an accurate finished sequence, but multiple sequencing platforms or paired-end libraries are often required to achieve full genome coverage. In this study, we demonstrated a method to assemble complete bacterial genome sequences by integrating shotgun Roche 454 pyrosequencing with optical whole genome mapping (WGM). The whole genome restriction map (WGRM) was used as the reference to scaffold <i>de novo</i> assembled sequence contigs through a stepwise process. Large <i>de novo</i> contigs were placed in the correct order and orientation through alignment to the WGRM. <i>De novo</i> contigs that were not aligned to WGRM were merged into scaffolds using contig branching structure information. These extended scaffolds were then aligned to the WGRM to identify the overlaps to be eliminated and the gaps and mismatches to be resolved with unused contigs. The process was repeated until a sequence with full coverage and alignment with the whole genome map was achieved. Using this method we were able to achieved 100% WGRM coverage without a paired-end library. We assembled complete sequences for three distinct genetic components of a clinical isolate of <i>Providencia stuartii:</i> a bacterial chromosome, a novel <i>bla</i>_NDM-1 plasmid, and a novel bacteriophage, without separately purifying them to homogeneity.</p></div

Detection of Bacterial <em>16S</em> rRNA and Identification of Four Clinically Important Bacteria by Real-Time PCR

<div><p>Within the paradigm of clinical infectious disease research, <em>Acinetobacter baumannii</em>, <em>Escherichia coli</em>, <em>Klebsiella pneumoniae</em>, and <em>Pseudomonas aeruginosa</em> represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial <em>16S</em> rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of <em>A. baumannii</em>, <em>E. coli</em>, <em>K. pneumoniae</em>, and <em>P. aeruginosa</em>. All primers were tested for specificity <em>in vitro</em> against 50 species of Gram-positive and –negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the <em>uidA</em> gene in <em>E.coli</em>, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.</p> </div