Rapid identification of blaKPC-possessing Enterobacteriaceae by PCR/electrospray ionization-mass spectrometry (2010)

Sir,

The spread of blaKPC genes responsible for the production of KPC carbapenemases among the family of Enterobacteriaceae (in particular Klebsiella pneumoniae species) is reaching alarming rates in America, Europe and parts of Asia.1 So far, nine different blaKPC genes (i.e. blaKPC-2 to blaKPC-10) encoding enzymes with very similar kinetic characteristics have been reported.1,2

Production of KPC carbapenemases confers resistance to all classes of β-lactams including those combined with standard β-lactamase inhibitors. In addition, isolates producing this enzyme are frequently co-resistant to quinolones, aminoglycosides and occasionally to colistin and fosfomycin.1,3,4 As a result, the rapid identification of blaKPC-possessing Enterobacteriaceae has serious implications for the outcome of infected patients. However, detection of blaKPC-possessing isolates presents a significant challenge for clinical laboratories.5,6 Therefore, a rapid and accurate system to identify these life-threatening pathogens would be welcome.

The PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) technology is a promising genotyping system that possesses high multiplexing capacity and can be used for detecting different genes (e.g. resistance traits and/or virulence factors) present in a single strain. This system can also detect single nucleotide polymorphisms including mutations corresponding to changes in existing amino acids. Primers designed for PCR/ESI-MS yield amplicons that are analytically characterized by high-performance MS and base composition analysis, which is expressed as the relative proportions of A, C, G and T.7 Previously, we demonstrated that PCR/ESI-MS could accurately detect mutations in gyrA and parC genes of quinolone-resistant Acinetobacter baumannii isolates.8 In the present work we evaluated the performance of the T5000™ Biosensor System (Ibis Biosciences Inc., Carlsbad, CA, USA), a PCR/ESI-MS platform,7 for the detection and identification of blaKPC genes among Enterobacteriaceae.

One hundred and ten previously characterized Enterobacteriaceae isolates (82 K. pneumoniae, 27 Escherichia coli and 1 Enterobacter cloacae) were used.3,4,9,10 In particular, there were 69 clinical isolates possessing a blaKPC gene (35 blaKPC-2 and 34 blaKPC-3)3,4,9 and 5 E. coli DH10B (Invitrogen Corporation, Carlsbad, CA, USA) control strains constructed in our laboratory carrying blaKPC-4, blaKPC-5, blaKPC-6, blaKPC-7 or blaKPC-8 in the pBC SK(+) vector. The remaining blaKPC-negative strains (17 K. pneumoniae and 17 E. coli isolates) were multidrug-resistant strains that have been described previously.10 ATCC control strains E. coli 25922 and K. pneumoniae 700603 were also used.

DNA was obtained by suspending two or three colonies of each test isolate grown on blood-agar plates in 500 µL of nuclease-free water (USB, Cleveland, OH, USA) and heating at 90°C for 10 min using a dry bath incubator (Fisher Scientific, Pittsburgh, PA, USA). Samples were spun at 10 000 rpm for 10 min and the resulting supernatant was diluted 1:100 with nuclease-free water. Aliquots of 10 µL were directly added to each of the 96 wells of the KPC Resistance Assay Kit plate (Ibis Biosciences Inc.) to a total of 24 samples per plate. Additional reagents were not necessary because each well contained 40 µL of a pre-loaded mixture consisting of buffer, dNTPs, enzyme and primers. In its current format this research kit uses primers designed to detect all known genes encoding KPC variants and to differentiate blaKPC-3 from blaKPC-4/blaKPC-5 and other KPC types. A primer pair specific for the valyl-tRNA synthetase (valS) housekeeping gene has also been included for species identification of Enterobacteriaceae.7 The primer pairs used in the kit are reported in Table 1. 



Table 1

Primers pairs used by the KPC Resistance Assay Kit to obtain the specific blaKPC amplicons analysed by ESI-MSa



Plates were incubated in the Mastercycler pro S Thermal Cycler (Eppendorf, Hauppauge, NY, USA) with the following PCR cycling conditions: 95°C for 10 min; 8 cycles of 95°C for 30 s, 48°C for 30 s and 72°C for 30 s; 37 cycles of 95°C for 15 s, 56°C for 20 s and 72°C for 20 s; 72°C for 2 min; and 99°C for 20 min (total, 2 h and 21 min). Plates were then loaded onto the automated T5000™ Biosensor System for analysis and interpretation of PCR amplicons as previously reported (total, 2 h and 30 min for one plate).7

Our results show that the T5000™ System correctly identified all 74 blaKPC-possessing strains; false-positive results were not observed (sensitivity and specificity of 100%). In particular, all blaKPC-2- and blaKPC-3-possessing strains were correctly reported. Notably, we were also able to identify all five KPC-positive E. coli DH10B control isolates, but we were not able to discriminate between blaKPC-6-, blaKPC-7- and blaKPC-8-containing isolates [these strains were reported as KPC-2 positive (for blaKPC-6) or KPC-3 positive (for blaKPC-7 and blaKPC-8)]. Finally, all tested strains were correctly identified at the species level.

In conclusion, the results of the present study show that the T5000™ System is an accurate tool to detect blaKPC-possessing Gram-negative isolates. Due to its rapid performance, the PCR/ESI-MS-based platform could be used in the clinical setting to improve the outcome of infected patients and could also be used to perform epidemiological and infection control studies where large collections of isolates need to be rapidly characterized.