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

Byline: Kwang Seung Park, Jung Hun Lee, Moonhee Park, Asad Mustafa Karim and Sang Hee Lee

ABSTRACT

Objective: Fast detection of b-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-scale 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-scale blaFinder detected all bla genes identified by the provider using microarray and multiplex PCR. In one of the Escherichia coli test isolates, a bla DHA-1 gene was detected using the multiplex PCR assay but it was not detected using the large-scale blaFinder.

Conclusion: The truncation of a bla gene is an important reason for an efficient molecular diagnostic method (large-scale blaFinder) not to detect the bla gene.

KEY WORDS: b-Lactamase (bla) gene, Large-scale detection, Molecular diagnosis, Minimizing antibiotic resistance.

INTRODUCTION

The development of fast and accurate diagnostic methods to detect antibiotic resistance genes is needed to minimise antibiotic resistance.b-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 b-lactam antibiotics through the acquisition of genes that encode b-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 b-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 b-lactam resistance, as b-lactam resistance can increase due to inappropriate b-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 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 blaDHA-1 gene was detected using the multiplex PCR assay designed by Perez-Perez and Hanson1 but it was not detected using the largescale blaFinder (Fig.1A and B).

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 (DblaDHA-1) lacking a 3' (or 5') end sequence in the E. coli test isolate.

DISCUSSION

The previous study showed a DblaDHA-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 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 DblaDHA-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 DblaDHA-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 DblaDHA-1 gene in the E. coli test isolate. That is because a truncated bla gene does not show any antibiotic resistance.

ACKNOWLEDGMENTS

We acknowledge financial supports of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (No.2016R1C1B2010308); and the Marine Biotechnology Program (No. 20150581, Development of Technology for Biohydrogen Production using Hyperthermophilic Archaea) funded by the Ministry of Oceans and Fisheries in Republic of Korea.

Declaration of interests: None.

REFERENCES

1. Lee JH, Park KS, Karim AM, Lee C-R, Lee SH. How to minimize antibiotic resistance. Lancet Infect Dis. 2016;16(1): 17-18. doi: 10.1016/S1473-3099(15)00467-3.

2. Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC b-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol. 2002;40(6): 2153-2162. doi: 10.1128/JCM.40.6.2153-2162.2002.

3. Dallenne C, Da Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important b-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010;65(3): 490-495. doi: 10.1093/jac/dkp498.

4. Ellem J, Partridge SR, Iredell JR. Efficient direct extended-spectrum b-lactamase detection by multiplex real-time PCR: accurate assignment of phenotype by use of a limited set of genetic markers. J Clin Microbiol. 2011;49(8): 3074-3077. doi: 10.1128/JCM.02647-10.

5. Monteiro J, Widen RH, Pignatari AC, Kubasek C, Silbert S. Rapid detection of carbapenemase genes by multiplex real-time PCR. J Antimicrob Chemother. 2012;67(4): 906-909. doi: 10.1093/jac/dkr563.

6. Leinberger DM, Grimm V, Rubtsova M, Weile J, Schroppel K, Wichelhaus TA, Knabbe C, Schmid RD, Bachmann TT. Integrated detection of extended-spectrum-b-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes. J Clin Microbiol. 2010;48(2): 460-471. doi: 10.1128/JCM.00765-09.

7. Barisic I, Schoenthaler S, Ke R, Nilsson M, Noehammer C, Wiesinger-Mayr H. Multiplex detection of antibiotic resistance genes using padlock probes. Diagn Microbiol Infect Dis. 2013;77(2): 118-125. doi: 10.1016/j. diagmicrobio.2013.06.013.

8. Grimm V, Ezaki S, Susa M, Knabbe C, Schmid RD, Bachmann TT. Use of DNA microarrays for rapid genotyping of TEM b-lactamases that confer resistance. J Clin Microbiol. 2004;42(8): 3766-3774. doi: 10.1128/JCM.42.8.3766-3774.2004.

9. Lee JJ, Lee JH, Kwon DB, Jeon JH, Park KS, Lee C-R, Lee SH. Fast and accurate large-scale detection of b-lactamase genes conferring antibiotic resistance. Antimicrob Agents Chemother. 2015;59(10): 5967-5975. doi: 10.1128/ AAC.04634-14.

10. Ho PL, Lo WU, Yeung MK, Lin CH, Chow KH, Ang I, Tong AH, Bao JY, Lok S, Lo JY. Complete sequencing of pNDM-HK encoding NDM-1 carbapenemase from a multidrug-resistant Escherichia coli strain isolated in Hong Kong. PLoS One. 2011;6(3): e17989. doi: 10.1371/journal.pone.0017989.
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Publication:Pakistan Journal of Medical Sciences
Date:Oct 31, 2016
Words:1445
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