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.2-8 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-scaleblaFinder) 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 large-scaleblaFinder 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 large-scaleblaFinder 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 blaDHA-1 gene was detected using the multiplex PCR assay designed by Perez-Perez and Hanson1 but it was not detected using the large-scaleblaFinder (Fig.1A and B).

Fig.1

PCR assays to detect a truncated blaDHA-1 gene using an Escherichia coli test isolate (lane 1 of each Figure), E. coli E07-10537 (a blaDHA-1 positive isolate; lane 2 of each Figure), and a blaDHA-1 negative Providencia stuartii isolate (lane 3 of each Figure). (A) Simplex PCR assays using a primer pair (DHAMF and DHAMR) used by the method of Perez-Perez and Hanson (1). Two same bands (405 bp) were detected in the E. coli test isolate and E. coli E07-10537. (B) Simplex PCR assays using a primer pair (DHA(AmpC-2) type-F and DHA(AmpC-2) type-R) used by the large-scaleblaFinder. Only one band (881 bp) was shown in E. coli E07-10537. (C) Simplex PCR assays using a primer pair (DHAMF and DHA(AmpC-2) type-R). Only one b and (642 bp) was detected in E. coli E07-10537. (D) Simplex PCR assays using a primer pair (DHA(AmpC-2) type-F and DHAMR). Two same bands (644 bp) were detected in the E. coli test isolate and E. coli E07-10537. (E) Schematic representation of the DNA sequences surrounding a truncated blaDHA-1 gene (ΔblaDHA-1) in E. coli 271 (Ho et al. (10)) and a newly designed primer pair. Each nucleotide position of ΔblaDHA-1 and each primer were shown in parenthesis. 345 bp (position: 796 to 1140) of blaDHA-1 sequence were missing at 3’ end. (F) Simplex PCR assays using a newly designed primer pair (trpF-F and DHA type-R). Only one band (734 bp) was shown in the E. coli test isolate. M1 (size marker), 100 bp DNA ladder (Biosesang, Korea).

To resolve this issue, simplex PCR assays9 were performed for the detection of blaDHA-1 gene using the Escherichia coli test isolate, E. coli E07-10537,9 and a blaDHA-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-scaleblaFinder (Fig.1C and D). The nucleotide position of the primer pair used by Perez-Perez and Hanson2 is 258-662. However, the nucleotide position of the primer pair used by the large-scaleblaFinder is 19-899. The results suggest that there is a truncated blaDHA-1 (ΔblaDHA-1) lacking a 3’ (or 5’) end sequence in the E. coli test isolate.

DISCUSSION

The previous study showed a ΔblaDHA-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’-ATGCCCGCGAAAATCAAGATTTG-3’; and DHA type-R, 5’-CAAAGCCAGTATGCGTACGG-3’) to know the exact truncated blaDHA-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 blaDHA-1 sequence were missing at 3’ end. The total sizes of ΔblaDHA-1 and blaDHA-1 were 795 bp and 1140 bp, respectively.9,10 Therefore, the efficient molecular diagnostic method (large-scaleblaFinder) could not detect the ΔblaDHA-1 gene in the E. coli test isolate. Because a truncated bla gene does not show any antibiotic resistance, the large-scaleblaFinder 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-scaleblaFinder) 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 ΔblaDHA-1 gene in the E. coli test isolate. That is because a truncated bla gene does not show any antibiotic resistance.