Rapid and simultaneous detection of the chlorhexidine and mupirocin resistance genes qacA/B and mupA in clinical isolates of methicillin-resistant Staphylococcus aureus.

We describe a real-time PCR-based assay capable of simultaneously detecting femA (Staphylococcus aureus-specific), mecA (methicillin resistance), qacA/B (chlorhexidine tolerance), and mupA (high-level mupirocin resistance) from bacterial cells in less than 90 minutes. The assay was validated with 1968 clinical MRSA submitted to a surveillance network.

Emergence of colistin-resistance in extremely drug-resistant Acinetobacter baumannii containing a novel pmrCAB operon during colistin therapy of wound infections.

BACKGROUND: Colistin resistance is of concern since it is increasingly needed to treat infections caused by bacteria resistant to all other antibiotics and has been associated with poorer outcomes. Longitudinal data from in vivo series are sparse. METHODS: Under a quality-improvement directive to intensify infection-control measures, extremely drug-resistant (XDR) bacteria undergo phenotypic and molecular analysis. RESULTS: Twenty-eight XDR Acinetobacter baumannii isolates were longitudinally recovered during colistin therapy. Fourteen were susceptible to colistin, and 14 were resistant to colistin. Acquisition of colistin resistance did not alter resistance to other antibiotics. Isolates had low minimum inhibitory concentrations of an investigational aminoglycoside, belonged to multi-locus sequence type 94, were indistinguishable by pulsed-field gel electrophoresis and optical mapping, and harbored a novel pmrC1A1B allele. Colistin resistance was associated with point mutations in the pmrA1 and/or pmrB genes. Additional pmrC homologs, designated eptA-1 and eptA-2, were at distant locations from the operon. Compared with colistin-susceptible isolates, colistin-resistant isolates displayed significantly enhanced expression of pmrC1A1B, eptA-1, and eptA-2; lower growth rates; and lowered fitness. Phylogenetic analysis suggested that colistin resistance emerged from a single progenitor colistin-susceptible isolate. CONCLUSIONS: We provide insights into the in vivo evolution of colistin resistance in a series of XDR A. baumannii isolates recovered during therapy of infections and emphasize the importance of antibiotic stewardship and surveillance.

Bacterial peritonitis due to Acinetobacter baumannii sequence type 25 with plasmid-borne new delhi metallo-ß-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 whole-genome 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 >99% homology to pNDM-BJ02. The isolate carried numerous other antibiotic resistance genes, including the 16S methylase gene, armA.

Detection of New Delhi metallo-ß-lactamase (encoded by blaNDM-1) in Acinetobacter schindleri during routine surveillance.

A carbapenem-resistant Alcaligenes faecalis strain was isolated from a surveillance swab of a service member injured in Afghanistan. The isolate was positive for bla(NDM) by real-time PCR. Species identification was reevaluated on three identification systems but was inconclusive. Genome sequencing indicated that the closest relative was Acinetobacter schindleri and that bla(NDM-1) was carried on a plasmid that shared >99% identity with one identified in an Acinetobacter lwoffii isolate. The isolate also carried a novel chromosomally encoded class D oxacillinase.

Rapid and simultaneous detection of blaKPC and blaNDM 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.

Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR.

Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa 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 16S 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 A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro 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 uidA gene in E.coli, 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.