LETTER
The worldwide prevalence of infections due to Gram-negative bacteria coresistant to extended-spectrum cephalosporins and carbapenems is worrisome. For these organisms, polymyxins are considered the last-resort antibacterials. Thus, the recent emergence of the plasmid-mediated colistin resistance genes mcr-1, mcr-2, and mcr-3 has become an alarming public health issue (1, 2) that urges rapid and reliable detection methods (3–6).
In this work, we evaluated the performance of the new commercial CT103XL microarray system (Check-Points). In ∼6.5 h (starting from culture strains) and for a cost ranging from 50 to 85€, this platform allows the detection of mcr-1 (including the variants from mcr-1.2 to mcr-1.7) and mcr-2 determinants. Along with them, clinically important class A, B, C, and D β-lactamase (bla) genes are simultaneously detected, as in the former version (CT103XL) that has demonstrated an overall accuracy of >94% (7).
We tested 106 Enterobacteriaceae isolates of human, animal, and food origin consisting of 80 strains of Escherichia coli, 14 of Klebsiella pneumoniae, and 12 of other species (Table 1). Most isolates were phenotypically (e.g., MICs for colistin) and molecularly (i.e., PCR/sequencing of mcr-1 and mcr-2 and bla genes or whole-genome sequencing) characterized in previous studies (4, 8–11) (see also Table S1 in the supplemental material). Sixty-one strains (57.5%) were resistant to colistin (12), of which 30 were positive for mcr-1 (including one positive for mcr-1.2) and two were positive for mcr-2. This group of 32 isolates (30.2%) included 7 blaESBL and one blaCMY-2 carrier, as well as 24 without bla or possessing only blaTEM-1-like. The other 74 mcr-1- and mcr-2-negative strains included 32 carbapenemase- and 24 extended-spectrum β-lactamase (ESBL)- and/or pAmpC-producing strains (Table S1).
Origin, resistance genes, and accuracy in detecting mcr-1 and mcr-2 of the new CT103XL microarray for the 106 tested strains
Total bacterial DNA was rapidly extracted from fresh cultured bacteria using Chelex-100 resin (Sigma-Aldrich). In the case of an unclear result, a new extraction was performed using the High Pure PCR template preparation kit (Roche). Then, microarray analyses were performed according to the manufacturer's instructions. The new CT103XL uses a multiplex ligation detection reaction. In brief, each probe consists of two arms containing target gene-specific sequence, a universal primer binding site, and a ZIP code for the hybridization (Fig. S1A). Ligated and amplified probes are hybridized to the microarray, visualized using biotin label incorporated into the primer, and automatically interpreted by software (13).
The mcr-1 and mcr-2 genes were correctly detected by the new array system in all 32 isolates (Table 1). In particular, the 30 mcr-1 carriers displayed positive reactions with all three new hybridization spots (Fig. S1B), corresponding to mcr-1 detection. On the other hand, both mcr-2-harboring strains displayed positive results with two of the three spots, revealing mcr-2 (Fig. S1C). Two microarray analyses had to be repeated due to inadequate template concentration. The second run, performed with new DNA extracts, yielded correct results (Table S1). Moreover, all 74 strains lacking mcr-1 and mcr-2 resulted negative after microarray analyses. Therefore, the new CT103XL platform showed 100% accuracy in identifying mcr-1 and mcr-2. We also noted that all bla genes were correctly identified (Table S1), thereby confirming previous validations (7, 14, 15).
This work demonstrated the ability and accuracy of the new CT103XL array to simultaneously identify mcr-1 and mcr-2 and clinically important bla genes. This strengthened platform represents therefore an essential tool for clinical laboratories, especially for the rapid characterization of large collections of multidrug-resistant Gram-negative bacteria, providing essential epidemiological data. The test is unable to detect the recently described mcr-3 (only 45% and 47% identity to mcr-1 and mcr-2, respectively) (2), but the flexibility of the microarray allows easy and rapid updating of the platform to include novel and emerging antimicrobial resistance traits.
ACKNOWLEDGMENTS
This work was supported by the Swiss National Science Foundation (SNF grant no. 153377 to A.E.). Odette J. Bernasconi is a Ph.D. student (2015 to 2018) supported by the Hans Sigrist Foundation (Bern, Switzerland).
A.K. is an employee of Check-Points. The new CT103XL microarrays used in the present study were provided for free by Check-Points to A.E.
We thank Surbhi Malhotra-Kumar (University of Antwerp, Wilrijk, Belgium) for donating the mcr-2-positive E. coli strain.
FOOTNOTES
- Accepted manuscript posted online 2 August 2017.
Supplemental material for this article may be found at https://doi.org/10.1128/JCM.01056-17 .
- Copyright © 2017 American Society for Microbiology.
