#211 |  ASM MICROBE 2018

Rapid Detection of OXA β-Lactamases by Multiplex Real-Time PCR

S. Cossette, M. P. Torres, J. Lechner, L. Porter, C. Connelly, M. Sobansky

Streck Research and Development-Molecular Technology Division, La Vista, NE

ABSTRACT

BACKGROUND

Background: The OXA β-lactamases have evolved to metabolize cephalosporins and carbapenems in addition to the penicillins, making them a growing problem when selecting effective antibiotic therapies. These enzymes are often associated with Acinetobacter spp., but due to the mobility of these genes, other organisms have acquired resistance to this class of enzymes, facilitating the spread of this type of antibiotic resistance. As such, assays that identify these resistance mechanisms are needed for faster detection of resistance-associated genes. These data can be used to supplement phenotypic test results and promote improved antimicrobial stewardship and surveillance. In this study, we describe a multiplex real-time PCR assay that successfully discriminates 6 genetically similar OXA β-lactamase gene families and utilizes a external positive control that fully mimics a patient sample.


Methods: The NCBI GenBank database was used to identify sequences for each OXA gene family. Sequences for each of the 6 groups were aligned to identify genetic variation between each OXA group, which guided primer and probe design to improve discrimination of each OXA family within the multiplex PCR reaction. A custom 2X qPCR MasterMix was run with the Streck ARM-D kit thermal cycling conditions on the BioRad CFX96 Real-Time PCR System for the multiplex reactions. An internal control (IC), which targets a conserved region in Gram-negative bacteria, was included to reduce false-negative results 1,2. External positive control samples containing several of the targets amplified by this kit were prepared via proprietary methods and run on the kit in conjunction with a positive control and clinical isolates.


Results: Together, the 6 oligo sets in this assay amplify a total of 224 OXA-like variants without cross reactivity between the OXA subgroups. Positive samples were identified within the first 22 cycles of PCR. Sensitivity and specificity for the control DNA tested in this assay was greater than or equal to 95% in each case. The external positive controls were positive for the expected targets, and performed comparably to the clinical isolates.


Conclusions: β-lactamases are a major mechanism of antibiotic resistance in Gram-negative bacteria, which continues to threaten health care facilities by reducing the available treatment options. Because there are many genes associated with antibiotic resistance, it is critical that tests such as these are developed to comprehensively detect these mechanisms. The assays described here provide a rapid detection strategy for genotypic monitoring of oxacillinase-based antibiotic resistance in Gram-negative bacteria. Inclusion of an external positive control allows the entire analytical process for a clinical specimen to be monitored during testing. More rapid identification of these genes provides an added tool to improve antibiotic stewardship practices and active surveillance of resistance mechanisms.

The OXA Real-Time PCR assay was developed to address the continual need to improve resistance testing and monitoring.  The OXA-like gene families detected by this assay are 6 of the OXA families that are as classified as Carbapenemase Hydrolyzing Class D β-lactamases (CHDL) which are clinically significant for their ability to produce resistance to antibiotics of last resort. This is a rapidly growing class of β-lactamase enzymes with more than 500 reported enzymes to date 1,2

Figure 1: Streck's ARM-D Kit

RESULTS

Data in Figures 2-5 illustrates the amplification of 6 OXA β-lactamase gene families detected by the OXA Real-Time PCR assay, and the comparable performance of external positive controls to clinical isolates.  The assay correctly identified the 17 isolates that carried one or more OXA β-lactamase genes, and 4 additional isolates were identified as positive that will be further characterized by sequencing, a total of 58 isolates were tested.  The assay also correctly identified the resistance genes carried by the four external positive control specimens.  As demonstrated in a previous study, DNA extracted from cells stabilized using this methodology produced Cq values that correlated to the number of cells in the extraction 5


Figure 2: Representative data generated by the internal control (IC) utilized in both of the OXA Real-Time PCR mixes.

Figure 4: Representative data generated by the OXA Real-Time PCR Mix #1.

Table 2: Summary of results. Average quantification cycle (Cq) values ± the standard deviation are shown for each target source across each of the targeted gene families. The data set (n) in each of the Average Cq columns represents the number of PCR reactions used to generate these values, all specimens and controls were run in duplicate. 

*Four of the isolates that generated positive results are omitted from these calculations due to pending confirmatory testing. 

Table 3: PCR cycling Conditions used for the OXA Real-Time PCR Assay 

MATERIALS & METHODS

Clinical isolates that had been previously characterized by singleplex PCR and/or sequencing were used to evaluate the performance of the assay. The Qiagen® DNeasy® Blood and Tissue Kit was used to extract nucleic acid from a set of overnight bacterial cultures (n=58) per the manufacturer’s instructions. A subset of these isolates carried various OXA β-lactamase resistance genes.
 The instructions for use from the Streck ARM-D kits was used to run the OXA Real-Time PCR assay on the BioRad CFX96 Real-Time PCR System. Each 25 μL PCR reaction was comprised of 12.5 μL of a custom 2x qPCR master mix, 2.5 μL of a 10x oligo mix, 9 μL of molecular grade water, and 1 μL of nucleic acid
6,7

SUMMARY AND CONCLUSIONS

  • Identifies and differentiates 6 OXA β-lactamase gene families with 100% sensitivity and specificity.
  • Can detect up to 224 OXA β-lactamase allelic variants within the described gene families. 
  • One of the first assays available to detect and discriminate OXA carbapenemases.
  • May promote improved antimicrobial resistance stewardship through reduced use of inappropriate antibiotics.
  • The external positive control ensures reliable and consistent analytical performance in the clinical setting by mimicking patient specimens.

REFERENCES & ACKNOWLEDGMENTS

  1. Poirel L, Naas T, Nordmann P. Diversity, Epidemiology, and Genetics of Class D β-Lactamases . Antimicrobial Agents and Chemotherapy. 2010;54(1):24-38. doi:10.1128/AAC.01512-08.
  2. Vázquez-Ucha JC, Maneiro M, Martínez-Guitián M, et al. Activity of the β-Lactamase Inhibitor LN-1-255 against Carbapenem-Hydrolyzing Class D β-Lactamases from Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy. 2017;61(11):e01172-17. doi:10.1128/AAC.01172-17.
  3. Evans BA, Amyes SGB. OXA β-Lactamases. Clinical Microbiology Reviews. 2014;27(2):241-263. doi:10.1128/CMR.00117-13.
  4. Antunes NT, Fisher JF. Acquired Class D β-Lactamases. Antibiotics. 2014;3(3):398-434. doi:10.3390/antibiotics3030398.
  5. Soabnsky M.R., Porter L.R. Development of Accurate and Reliable Full-Process Controls that Mimic Patient Samples for Molecular Diagnostic Testing. Poster Presentation ASM Microbe 2017.
  6. Streck. Streck ARM-D Kit, β-Lactamase (RUO). Ref. 350670-1. Nov. 2017.
  7. Streck. Streck ARM-D Kits (RUO) Data Acquisition & Analysis Guide – Bio-Rad® CFX96 Touch™ Real-Time PCR Detection System. Ref. 880107-1. Dec. 2017.
  8. QuickGene Series Application Guide. Genomic DNA extraction from Pseudomonas aeruginosa. No. 37.


We would like to thank Streck for support of this work. We acknowledge and thank Scott Whitney, Jeremiah Athmer, Michelle Prine and Drew Egger for their review of the poster. 

The poster titled Rapid Detection of OXA β-Lactamases by Multiplex Real-Time PCR  has been reformatted for digital consumption. 

Download original poster
Download original poster

Table 1: OXA β-lactamase gene families identified in each master mix of the OXA Real-Time PCR Assay. The same internal control (IC) is included in each master mix.

This assay was designed based on the format of the antimicrobial resistance monitoring detection (ARM-D) kits and  is capable of rapid identification and discrimination between the 6 OXA families. This information is valuable as resistance and reduced susceptibility for certain drugs varies between the different enzymes and may rely on the presence of addition resistance mechanisms 3,4. Existing assays are designed to identify the more common OXA β-lactamases, such as OXA-48, and may not be able to detect these clinically significant variants 2,4.  

In addition, the OXA Real-Time PCR assay was developed to incorporate external positive controls as a part of the test kit.  These controls are needed to meet the  requirements described by CAP, ISO, and the CFR for controls used in molecular diagnostic testing. Each of these entities describes specific guidelines to ensure consistent and reliable practices for patient testing 5. The external positive control included in the OXA Real-Time PCR assay contains stabilized microorganisms and mimics patient samples thereby monitoring the entire testing system as described by the regulatory agencies.

Figure 3: Direct comparison of amplicons generated by DNA extracted from fresh culture and from stabilized cells. The same bacterial isolate was used in both preparations.

Figure 5: Representative data generated by the OXA Real-Time PCR Mix #2.

Duplicate reactions were run for all specimens and controls.

Four Gram-negative organisms derived from clinical specimens (2 Acintobacter baumannii, 1 Acintobacter spp., and 1 Klebsiella pneumonia), containing various OXA β-lactamase resistance genes, were chemically stabilized in a cellular suspension according to proprietary procedures. These stabilized cells served as external positive controls for the OXA Real-Time PCR assay. DNA was extracted from each control using the QuickGene DNA tissue kit S and the QuickGene-810 system according to previously published procedures 8.

Two of the isolates used to prepare the external positive controls were also part of the set of clinical isolates evaluated by the assay allowing for a side-by-side comparison of the control with an unknown sample. The other control isolates contained different targets across both mastermixes. 

Amplicons generated by the clinical isolates were compared to the positive controls and used to determine the specificity and sensitivity of the assay based on the correct identification of specimens that carried resistance genes versus specimens that did not.

External Positive Control

Kit Positive Control

Clinical Isolates

OXA-48: External Positive Control

OXA-48: External Positive Control

IC: Clinical Isolates

IC: External Positive Control

Kit Positive Control

Clinical Isolates

Kit Positive Control

External Positive Control

Clinical Isolates

Kit Positive Control

External Positive Control

Clinical Isolates

Kit Positive Control

External Positive Control

Kit Positive Control

External Positive Control

Clinical Isolates

Kit Positive Control

External Positive Control

Clinical Isolates

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