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Identification of antibiotic resistance genes in Klebsiella pneumoniae isolates and metagenomic samples using real-time PCR arrays Matthew Fosbrink1, Geoffrey Wilt1, Liang Chen2, Barry Kreiswirth2, Vikram Devgan1

1QIAGEN Sciences Inc., Frederick, MD, 2Public Health Research Institute Center, New Jersey Medical School, UMDNJ Newark, NJ

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Abstract

Treatment of bacterial infections has become more difficult due to increasing rates of antibiotic resistance, highlighting the importance of prevention and surveillance. As effective surveillance activities are vital in determining the measures needed to control antibiotic resistance, new and rapid laboratory methods are necessary to facilitate this important effort. Real-time PCR methods have proven effective for the detection of antibiotic resistance genes and PCR array technology allows the detection of a large number of genes in a single PCR run. Therefore, in this study, an antibiotic resistance gene identification PCR array was developed that allows for rapid screening of a range of antibiotic resistance genes present in a sample. The PCR array contains 5′ hydrolysis probe assays (primer and dual-labeled probe sets) that uniquely target 87 antibiotic resistance genes. All assays exhibited low-end sensitivity between 5–80 copies and a linear dynamic range of at least 5 orders of magnitude. A pilot research study was performed on a collection of Klebsiella pneumoniae isolates to identify the diversity of resistance genes. The PCR results revealed that the SHV antibiotic resistance gene was present in all 14 K. pneumoniae isolates tested and 10 (71.4%) of the isolates were positive for non-ESBL targets SHV-156G/238G240E. One (7.1%) sample harbored the ESBL SHV-156D mutation and three (21.4%) isolates harbored the SHV-238S240E variant. To verify the results from the PCR array, a subset of the antibiotic resistance genes were analyzed by pyrosequencing. Results of pyrosequencing confirmed the presence of KPC, SHV-156G, SHV-156D, SHV-238G240E, SHV-238S240E, tetA, tetB, CTX-M-1/2 groups, AAC(6)-lb-cr, and aadA1 in the K. pneumoniae isolates. Since the gut is known to act as a reservoir for antibiotic resistance genes, a small-scale research study was performed on five stool samples isolated from healthy human adults using the antibiotic resistance gene identification PCR array. All five samples had ermB and mefA, and three of the samples were positive for tetA. In conclusion, PCR arrays can be effective and reliable tools for profiling antibiotic resistance genes from both isolates and metagenomic samples.

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Introduction

Antibiotic resistant strains of pathogenic bacteria are a growing worldwide health problem. To effectively combat the spread of difficult-to-treat bacterial infections, rapid surveillance methods for detection of antibiotic resistance genes is required to monitor both bacterial isolates and metagenomic samples. Additionally, identification of potential new sources for different antibiotic resistance genes is critical. Both of these goals require tools that can be used for profiling of antibiotic resistance genes from various types of samples. Real-time PCR has proven to be effective for the detection of antibiotic resistance genes. Using PCR array technology, simultaneous detection of 87 prevalent and important antibiotic resistance genes is possible and should prove to be an effective method for antibiotic resistance monitoring. This allows for a more comprehensive profiling of antibiotic resistance genes than is possible using individual PCR assays.

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Microbial PCR array method

Genomic DNA from Klebsiella pneumoniae isolates were extracted using a Promega Wizard® Genomic DNA Purification Kit. Genomic DNA from stool samples were extracted using a QIAGEN QIAamp® DNA Stool Mini Kit.

Each sample comprised 250 ng of genomic DNA from Klebsiella pneumoniae isolates or 500 ng of genomic DNA from stool samples. Samples were mixed with microbial qPCR probe mastermix and microbe-free water, and this mixture was then uniformly dispensed into a 96-well PCR plate containing dried-down primers and 5’-hydrolysis probes for each of the antibiotic resistance genes tested.

Each PCR plate was run on a Roche LightCycler 480 using the following cycling conditions:

After the PCR run, raw CT values were exported to the microbial qPCR analysis software to detect the presence of antibiotic resistance genes. The identification criteria were as follows: CT<32 was identified as positive, CT>35 was identified as negative and a 32<CT<35 was inconclusive. In addition, the control assay PPC (Positive PCR Control) had to have a CT=22±2 to show that the PCR instrument and mastermix performed properly and there were no PCR inhibitors in the sample.

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Antibiotic resistance gene screening microbial qPCR array

Figure 1. Layout of antibiotic resistance gene screening microbial qPCR array. The antibiotic resistance gene screening microbial qPCR array allows identification of different antibiotic resistance genes in a single PCR run. Each array contains controls such as Pan-Bacteria 1 and Pan-Bacteria 2 to detect total bacteria and ensure bacterial genomic DNA was added to the array. The control PPC (Positive PCR Control) confirms a positive PCR run and the absence of PCR inhibitors in the sample.

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Linearity and sensitivity of microbial qPCR assays

Figure 2. Linearity and sensitivity for each assay on the microbial antibiotic resistance gene qPCR array. Linearity and sensitivity were determined using synthetic templates over a 6-log serial dilution ranging from 1 copy to 1 million copies. The following are representative results for all the assays on the array. [A] Real-time amplification curves of the KPC antibiotic resistance gene qPCR assay. [B] A standard curve shows the primer efficiency equals 103% (calculated from slope=-3.3236) with a correlation coefficient of 0.9983, indicating optimum performance for the KPC qPCR assay.

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Identification of multi-drug antibiotic resistance in Klebsiella pneumoniae isolates

Figure 3. Screening of Klebsiella pneumoniae isolates using antibiotic resistance gene microbial qPCR array. 250 ng of genomic DNA, isolated from Klebsiella pneumoniae isolates, was loaded onto an antibiotic resistance gene microbial qPCR Array. PCR was performed and raw CT values were imported into the microbial qPCR array analysis spreadsheet. Positive + /negative - / inconclusive +/- result for each antibiotic resistance gene was determined by the analysis software using the identification criteria. The results show that different classes of antibiotic resistance genes may be present in the same Klebsiella pneumoniae isolate.

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Verification of specificity for PCR array by pyrosequencing

(Legend on next slide)

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Figure 4. Pyrosequencing verification for the specificity of the antibiotic resistance gene microbial qPCR array results from Figure 3. Pyrosequencing assays were designed to detect for the presence and sequence of SHV-156G, SHV-156D, SHV-238G240E, SHV-238S240K, SHV-238S240E, SHV-238G240K, ermB, mefA, tetA, tetB, CTX-M-1 group, CTX-M-2 group, AAC(6)-lb-cr, and aadA1. For each Klebsiella pneumoniae isolate, results from the antibiotic resistance gene microbial PCR array were confirmed by pyrosequencing. Representative pyrograms for [A] SHV-156G, SHV-238/240, and [B] KPC and the CTX-M-1 group are shown above. For SHV variants, the microbial qPCR array was able to reliably distinguish single nucleotide polymorphisms occurring at different sites.

Verification of specificity for PCR array by pyrosequencing cont.

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Screening of gut microbiota for presence of antibiotic resistance genes

Figure 5. Several antibiotic resistance genes are prevalent in stool samples. Stool samples from five healthy adults were collected and genomic DNA was isolated. 500 ng of genomic DNA from each stool sample were analyzed for presence of antibiotic resistance genes using the antibiotic resistance gene microbial qPCR array. ErmB, mefA, and tetA were found in all or most of the stool samples tested, showing that they may be highly prevalent in the gut. These antibiotic resistance genes have been reported to be isolated from bacterial strains originating from food, suggesting a possible source of origin. This highlights the importance of increased surveillance of antibiotic resistance reservoirs to identify potential sources of antibiotic resistant bacteria.

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Summary

• Microbial qPCR arrays are a collection of sensitive and specific qPCR assays for the detection of antibiotic resistance genes from both bacterial isolates and metagenomic samples.

• Detection of 87 antibiotic resistance genes can be performed simultaneously in one 3-hour PCR run.

• The antibiotic resistance gene microbial PCR array is an effective tool for monitoring potential outbreaks of antibiotic resistant bacteria and identifying new sources of antibiotic resistant genes.

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