Why cannot a β-lactamase gene be detected using an efficient molecular diagnostic method?

Objective: Fast detection of β-lactamase (bla) genes can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We have evaluated a further refinement of our fast and accurate molecular method, developed to overcome these limitations, using clinical isolates. Methods: We have recently developed the efficient large-scale bla detection method (large-scaleblaFinder) that can detect bla gene types including almost all clinically available 1,352 bla genes with perfect specificity and sensitivity. Using this method, we have evaluated a further refinement of this method using clinical isolates provided by International Health Management Associates, Inc. (Schaumburg, Illinois, USA). Results were interpreted in a blinded manner by researchers who did not know any information on bla genes harbored by these isolates. Results: With only one exception, the large-scaleblaFinder detected all bla genes identified by the provider using microarray and multiplex PCR. In one of the Escherichia coli test isolates, a blaDHA-1 gene was detected using the multiplex PCR assay but it was not detected using the large-scaleblaFinder. Conclusion: The truncation of a blaDHA-1 gene is an important reason for an efficient molecular diagnostic method (large-scaleblaFinder) not to detect the bla gene.


INTRODUCTION
The development of fast and accurate diagnostic methods to detect antibiotic resistance genes is needed to minimise antibiotic resistance. 1 β-Lactam antibiotics are some of the most successful drugs used for the treatment of bacterial infections and represent roughly 65% of the total world market for antibiotics. 1 Therefore, resistance to β-lactam antibiotics through the acquisition of genes that encode β-lactamases is one of the most serious problems in Gram-negative pathogenic bacteria.
To date several molecular diagnostic methods of bla gene typing have been developed to detect the existence of β-lactamase (bla) gene(s) in clinical isolates. [2][3][4][5][6][7][8] These methods can detect only some (limited) bla genes. Because these methods cannot detect bla gene types including almost all clinically available bla genes, they cannot perfectly explain the results of the culture-based phenotypic tests. 9 This is a big problem in studying β-lactam resistance, as β-lactam resistance can increase due to inappropriate β-lactam use. To solve this problem, we have recently developed the efficient large-scale bla detection method ( large-scale blaFinder) that can detect bla gene types including almost all clinically available 1,352 bla genes with perfect specificity and sensitivity. 9

METHODS
We have evaluated a further refinement of this method using clinical isolates provided by International Health Management Associates, Inc. (Schaumburg, Illinois, USA), using the largescale blaFinder method. 9 Results were interpreted in a blinded manner by researchers who did not know any information on bla genes harbored by these isolates. With only one exception, the largescale blaFinder detected all bla genes identified by the  provider using microarray (Check-MDR CT101, Check-Points B.V., Wageningen, the Netherlands) and multiplex PCR. 2 In one of the Escherichia coli test isolates, a bla DHA-1 gene was detected using the multiplex PCR assay designed by Perez-Perez and Hanson 1 but it was not detected using the largescale blaFinder ( Fig.1A and B).
To resolve this issue, simplex PCR assays 9 were performed for the detection of bla DHA-1 gene using the Escherichia coli test isolate, E. coli E07-10537, 9 and a bla DHA-1 negative Providencia stuartii isolate.

RESULTS
Interestingly, in the E. coli test isolate, no band was detected using the reverse primer (DHA (AmpC-2) type-R) 9 used by the large-scale blaFinder ( Fig.1C and  D). The nucleotide position of the primer pair used by Perez-Perez and Hanson 2 is 258-662. However, the nucleotide position of the primer pair used by the large-scale blaFinder is 19-899. The results suggest that there is a truncated bla DHA-1 (Δbla DHA-1 ) lacking a 3' (or 5') end sequence in the E. coli test isolate.

DISCUSSION
The previous study showed a Δbla DHA-1 lacking a 3' end sequence (Fig.1E). 10 Based on the pNDM-HK sequence (HQ451074), we newly designed a primer pair (trpF-F, 5'-ATGCCCGCGAAAATCAA-GATTTG-3'; and DHA type-R, 5'-CAAAGCCAG-TATGCGTACGG-3') to know the exact truncated bla DHA-1 sequence in the E. coli test isolate (Fig.1E). Using these two primers, one band (734 bp) was detected in the test isolate (Fig.1F). Sequencing data of this band showed that 345 bp (position: 796 to 1140) of bla DHA-1 sequence were missing at 3' end. The total sizes of Δbla DHA-1 and bla DHA-1 were 795 bp and 1140 bp, respectively. 9,10 Therefore, the efficient molecular diagnostic method ( large-scale blaFinder) could not detect the Δbla DHA-1 gene in the E. coli test isolate. Because a truncated bla gene does not show any antibiotic resistance, the large-scale blaFinder has no problem for monitoring the emergence and dissemination of bla genes and minimizing the spread of resistant bacteria. Therefore, the truncation of a bla gene is an important reason for an efficient molecular diagnostic method not to detect the bla gene.

CONCLUSION
The efficient large-scale bla detection method ( large-scale blaFinder) is a useful test to detect bla gene types including almost all clinically available genes with perfect specificity and sensitivity, although the method could not detect the Δbla DHA-1 gene in the E. coli test isolate. That is because a truncated bla gene does not show any antibiotic resistance.