MagNA Pure System Application Note No. 3

Abstract

In May and June 2011, there was a significant increase in the number of cases of bloody diarrhea associated with hemolytic-uremic syndrome (HUS) caused by enterohemorrhagic Escherichia coli (EHEC). The majority of EHEC infected individuals lived in Germany, but significant numbers of infections have also been reported in France, Sweden, Russia and other European countries.

To detect the EHEC bacterium, the automated MagNA Pure 96 System for nucleic acid isolation and LightCycler®* Real-Time PCR Instrument (Roche Diagnostics) were used for high throughput examination of EHEC directly in stool samples.

Human stool samples were processed using the high throughput MagNA Pure 96 System to isolate high quality total genomic DNA. Targeting the Shiga toxin genes of enterohemorrhagic E. coli by the LightCycler®* Real-Time PCR instrument demonstrated that fecal samples can be used as starting material to detect EHEC infection, reducing the turnaround from days to hours.

March 2015

Ralf Bialek*, Olfert Landt**,

Markus Bollwein***, Udo Reischl***

* Labor Dr. Krause & Kollegen MVZ GmbH, Kiel, Germany

** TIB Molbiol GmbH, Berlin, Germany

*** Institute of Medical Microbiology and Hygiene,

University of Regensburg, Regensburg, Germany

Increased Throughput and Decreased Turnaround Time of enterohemorrhagic E. coli (EHEC) Extraction and Detection in Stool Samples through Use of MagNA Pure 96 System and LightCycler®* Instrument

*The LightCycler® 480 Instrument is for life science research only. Not for use in diagnostic prodcures.

2

Abstract continued

The fully automated robotic MagNA Pure 96 System used in this study isolates nucleic acids from 96 samples in approximately one hour. We have used an analysis assay containing two pairs of LightCycler® HybProbe probes for the LightCycler® 480 Instrument, detecting and distinguishing the Shiga toxin genes stx1 and stx2. The absence of E. coli intimin and enterohemolysin genes as well as a negative result for stx1 and a positive result for stx2 with a specific Tm of +71°C was characteristic for the epidemic HUSEC 41

Introduction

Shiga toxin-producing Escherichia coli (STEC) strains, also known as verotoxin-producing E. coli (STEC or VTEC) or enterohemorrhagic E. coli (EHEC) strains, are an important cause of food borne disease worldwide (1). Beginning in May 2011, Northern Germany experienced a severe out-break of EHEC-infections. These gram-negative bacteria are infected by bacteriophages and enabled to produce toxins. Two types of toxin coding genes have been described in EHEC: Shiga toxin 1 (stx1) which differs from the true Shiga toxin described in Shigella dysenteriae type 1 by one to seven amino acids, and Shiga toxin 2 (stx2) showing 60% homology to stx1.

Shiga toxins produced by E. coli can cause uncomplicated diarrhea, but also hemorrhagic colitis which can progress into hemolytic uremic syndrome (HUS), composed of a microangiopathic hemolytic anemia, thrombocytopenia and severe acute renal failure, requiring intensive care. For this reason, Shiga toxin-producing bacteria are also known as enterohemorrhagic E. coli (EHEC) (2). EHEC strains can also produce a plasmid coded hemolysin (blood cell destroy-ing toxin). Nearly 60% of EHEC strains belong to one of the three serogroups O157, O103 and O26 (in which the letter O indicates the lipopolysaccharide surface antigen and the number its type).

Every year approximately 1000 cases of EHEC infection are reported to German Health authorities. Infections are characterized by painful, bloody diarrhea of short duration. In 5% of cases, however, a hemolytic-uremic syndrome (HUS) occurs, requiring in 50% of these subjects temporal hemodialysis due to renal insufficiency, and in 50% of these cases, permanent dialysis. Usually children less than 5 years of age are affected, but elderly (> 60 years), and immunocompromised individuals are also at risk. Shiga toxin 2 is more often associated with disease complications.

In the recent outbreak, more than 3400 cases of EHEC infections were reported, mainly affecting young women between 20 to 50 years old, with a complication rate (HUS and/or neurologic symptoms) in more than 20%, and 39 deaths (3, RKI* 20.06.2011).

*Robert Koch Institute (RKI)

Currently, EHEC identification in the microbiology laboratory is a time consuming procedure: it is impossible to distinguish EHEC by morphological criteria from other E. coli, the most common bacterial species isolated from fecal specimens. Additionally, the number of EHEC bacteria shed with the stool is usually very low. Guidelines of the German Society of Hygiene and Microbiology** recommend the use of non-selective agars, such as MacConkey agar, for growing enterobacteriaceae, and testing the surface antigen O157, the most common EHEC in Germany. In addition, some colonies are selected for growth in an overnight enrichment broth, including mitomycin C to stress potential EHEC to produce verotoxins. The next day, verotoxin is measured in the supernatant using an enzyme-linked immunosorbent assay (EIA). Due to the low numbers of EHEC in stool samples, direct testing of verotoxins in feces is not recommended but overnight enrichment broth with stool can be performed. False negative results using these methods are known to occur.

**Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM)

EHEC is confirmed by isolating an E. coli colony producing verotoxin using EIA and/or carrying Shiga toxin 1 or 2 gene detected by PCR (4). In this recent outbreak, health authorities reported an unusual increase of HUS-cases, and identified E. coli O104:H4 strain producing Shiga toxin 2, but not Shiga toxin 1, as well as an Extended-Spectrum Beta-

strain. The parC gene was used as both an extraction control and inhibition control for these tests. The MagNA Pure 96 and LightCycler® used here to detect EHEC-infections performed accurate and sensitive tests. In the 181 samples examined, a sensitivity of 100% and a specificity of 98% were achieved. In contrast to routine microbiological procedures, PCR testing produced reliable data within hours.

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We used the automated MagNA Pure 96 System for extrac-tion of nucleic acids, combined with the LightCycler® 480 Instrument for PCR. To reduce workload, costs and time for EHEC detection, 181 fecal samples sent to our lab were screened for pathogenic microorganisms, including EHEC, the focus of this study.

Lactamase (ESBL, CTX-M-15 type) and fermenting sorbitol. This strain has an unusual combination of virulence factors of STEC/VTEC and enteroaggregative E. coli, EAggEC, with a special adhesion protein that attaches to intestinal cells (5). This new strain was later defined as HUSEC 41, sequence type ST678 (registered HUS syndrome causing E. coli strains, by the HUS reference laboratory).

if verotoxin positiveand/orand/or

and/or and/or

Result (for 181 samples)4–6 h

Result (for 181 samples)minimal 24 h; mean 40 h; in some cases up to 4 days

LightCycler® 480 System Blockcycler PCR | 4–6 h Blockcycler PCR | 4–6 h

Usually serological testing of E. coli-like colonies with anti-O157

Selective agar for ESBL-producing enterobacteriaceae

Enrichment broth containing mitomycin C

MacConkey Agar

Verotoxin testing by EIA | 3–4 h

Blockcycler PCR from enrichment broth sediment after DNA-extraction or boiling | 6–8 h

Subculture on ESBL- screening agar and/or MacConkey agar for Blockcycler PCR after overnight incubation

Subculture in enrichment broth for verotoxin testing after overnight incubation

Subculture in enrichment broth for verotoxin testing after overnight incubation

Collection of fecal specimen

Specimen handling | 1 h

MagNA Pure 96 System

Specimen handling | 2–4 h / overnight incubation | 16 h

Inoculation of

Routine bacteriological testing according to recommendations of Robert Koch-Institute and/or DGHM guidelines (German Society for Hygiene and Microbiology)

Figure 1: Comparison of time requirements to achieve results concerning EHEC in stool samples after entering the laboratory.

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Materials and Methods

MagNA Pure 96 template DNA extraction: Stool samples were collected in plastic containers without additives. Depending on consistency samples were mixed with up to 2.5 volumes of sterile 0.9% NaCl solution and homogenized by shaking. Then, 1 ml of each sample was transferred to a sterile reaction tube and centrifuged at 10,000 x g for 1 min to sediment corpuscular solid parts. Two hundred microliters of the supernatant were transferred to each well of the MagNA Pure 96 sample plate. Optional steps, described in the MagNA Pure 96 DNA and Viral NA Small Volume Kit package insert, such as DTT treatment, bacterial lysis, proteinase K handling or boiling, were not required.

For the MagNA Pure 96 System run, the new Pathogen Universal Protocol with a sample volume of 200 µl was chosen on the software menu. Elution volume was set to 100 µl. After samples were transferred to the instrument, prefilled kit reagents and disposables were loaded, according to the manufacturer’s recommendations. Fully automated robotic isolation of 96 samples was performed in approximately one hour.

LightCycler® 480 PCR and Melting Curve (Tm) analysis of amplicons:For the PCR analysis using the LightCycler® 480 Instrument, a consensus primer pair was used to amplify the E. coli Shiga toxin genes. Two pairs of fluorescently labeled LightCycler® HybProbe probes were used to detect stx1 and stx2, and to discriminate the known Shiga toxin gene variants (1). A crossing point (Cp) of <36 cycles was regarded as positive.

A negative result for stx1 (640 nm) and a positive result for stx2 (660 nm) with a specific Tm of +71°C, was found to be specific for the epidemic HUSEC 41 strain. The E. coli intimin (eae) and enterohemolysin A (hlyA) gene-specific PCR assays (1) were not performed, because these pathogenicity factors were found to be absent in the epidemic HUSEC 41 strain.

Five microliter aliquots of template DNA, purified using the MagNA Pure 96 Instrument, were added to each of the 15 µl LightCycler® FastStart DNA Master HybProbe master mixes (Roche Diagnostics), containing the corresponding primer and probe oligonucleotides (TIB Molbiol, Berlin).

The LightCycler® 480 Instrument was also used for semiquantitative determination of the parC gene (topoisomerase IV subunit of Enterobacteriaceae) using specific primers and LightCycler® FastStart DNA Master HybProbe master mix. Monitoring the detection of the parC gene in all samples tested served as a control for extraction and the absence of PCR inhibition.

Data for amplification and melting curve analysis

Amplification Curves

Cycles

Fluo

resc

ence

(49

8–64

0)

7.954-

7.154-

6.354-

5.554-

4.754-

3.954-

3.154-

2.354-

1.554-

0.754-

-0.046-

- - - - - - - - - - - - - - - - - - - - - -

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Amplification Curves

Cycles

Fluo

resc

ence

(49

8–66

0)

3.021-

2.721-

2.421-

2.121-

1.821-

1.521-

1.221-

0.921-

0.621-

0.321-

0.021-

- - - - - - - - -5 10 15 20 25 30 35 40 45

Figure 2a: Amplification curve of stx1. The two EHEC positive controls #345 (stx1+, stx2-) and #347 (stx1+, stx2+, pink curves) and one stool sample show the presence of stx1. The negative control (black line), and other samples as well as the control #346 (stx1-, stx2+) are negative, showing no signal.

Figure 2b. Amplification curve of stx2. The two EHEC positive controls #346 and #347 (pink curves) show the presence of stx2. The negative control (black line) and control #345 show no signal.

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Extraction Validation by Microbiology Culture:Feces were streaked on MacConkey and Brilliance-ESBL agar plates (Oxoid, Wesel, Germany) for overnight incuba-tion at 36°C. Another portion of the stool sample was used for inoculation of an enrichment broth containing mitomycin C (r-biopharm, Darmstadt, Germany). After overnight incubation the enrichment broth was centrifuged and 200 µl of the supernatant used in a verotoxin EIA (r-biopharm).

Isolates of E. coli grown on MacConkey or ESBL-screening agars were tested for verotoxin production directly or used for inoculation of the enrichment broth for verotoxin testing. Suspected EHEC strains were identified according to recommendations of reference laboratories (5). Non-targeted EHEC strains either showing different resistance pattern and/or carrying the Shiga toxin 1 gene detected by PCR were sent to the reference lab for further characterization.

Amplification Curves

Cycles

Fluo

resc

ence

(49

8–64

0)

6,505-

5,905-

5,305-

4,705-

4,105-

3,505-

2,905-

2,305-

1,705-

1,105-

0,505-

-0,095-

- - - - - - - - - -

5 10 15 20 25 30 35 40 45 50

Melting Peaks

Temperature (°C)

-(d/

dT)

Fluo

resc

ence

(49

8–66

0)

0.300-

0.210-

0.120-

0.030-

- - -

65 70

Results

Control PCR targeting the parC gene serving as extraction control, was positive in 178 of the 181 stool samples tested. This finding shows that DNA extraction was achieved in 98% of fecal specimens examined. The three negative parC samples were highly aqueous diarrhea samples which probably did not contain sufficient amounts of enterobacteriaceae in the sample material.

qPCR Data Compared to Routine Microbiology TestingReal-time PCR to detect the stx1/stx 2 genes using the LightCycler® 480 Instrument, amplified EHEC DNA in 36 samples and 142 samples were negative for stx1/stx2. Three samples without proof of sufficient DNA extraction, using the internal parC gene extraction control, were exclud-ed (see above). Of the 36 positive samples, 35 were positive

for the stx2 gene, and one was positive for the stx1 gene. These experimental results correlated with data obtained using microbiology testing. In 33 of the 36 positive samples the epidemic ESBL- and Shiga Toxin 2-producing EHEC strain was isolated, whereas a sporadic stx1 producing EHEC strain with different resistance pattern, not growing on ESBL-screening plates was isolated from the LightCy-cler® stx1 positive sample.

In the remaining two positive research samples using the LightCycler® System PCR neither EHEC was isolated nor verotoxin detected. In the other 142 fecal samples negative for stx1 and stx2 using the LightCycler® System PCR, neither EHEC was isolated nor verotoxin was found. None of the subjects found to be negative in these assays developed an

Figure 2c. Melting curve analysis of stx2 amplicons. A melting peak at +71°C indicates a positive result for the stx2 gene of the epidemic HUSEC 41 strain. Positive controls #346 and #347 show the melting peak, whereas the control #345 does not, as expected.

Figure 2d. Amplification curve of parC. All but 3 samples show amplification of the E. coli parC gene. Positive (pink curve) and negative controls (black line) showed the expected results. Since the three parC negative samples were high-ly aqueous diarrhea samples, they probably contained insufficient amounts of E. coli organisms to generate a positive PCR result for the parC target gene.

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Discussion

In an urgent situation, there is an inherent need to reduce hands-on time, a clear workflow for identifying positive sam-ples, and rapid screening with appropriate test controls. Total genomic DNA prepared directly from fecal specimens for PCR is a fast way to screen for pathogenic toxin genes of enterodiarrheagenic E. coli (3).

This study was undertaken as a proof-of-principle to examine whether automated systems are capable of identifying EHEC positive samples if DNA is extracted directly from the stool specimens. A single PCR-negative sample clearly does not exclude the possibility of an EHEC infection. However,

EHEC infection and/or HUS. For an overview, see Figure 3.Results continued

Time requirements for nucleic acid purification and PCRTo process 181 fecal samples, two purification runs were performed using the MagNA Pure 96 Instrument with 96 well plates; each run was completed within 58 minutes. The LightCycler® 480 Instrument was then used to perform two runs using 96 well plates. Fifty PCR amplification cycles were completed in 60 minutes. As described earlier (1),

discrimination between PCR-positive and PCR–negative samples was already possible after 30 cycles, i.e. within just 30 minutes. The combined MagNA Pure 96 and LightCycler® 480 System PCR workflow targeting the stx1 and stx2 genes detected 34 EHEC-positive stool samples out of the 181 samples examined in our laboratory, in 4 to 6 hours. These data are in accordance with routine microbio-logical testing requiring a mean time of 40 hours to detect verotoxin producing EHEC isolates in stool samples.

neither verotoxin screening by EIA nor PCR using DNA prepared from MacConkey agars were able to detect all positive EHEC cases in the most recent outbreak in Northern Germany (Bialek et al., manuscript in preparation). It cannot be overemphasized that detection of stx 1 or 2 gene in feces has to be further confirmed by conventional culture of EHEC and characterization of the Shiga toxin-producing isolate before an EHEC infection is proven. Nevertheless, our findings using automated extraction and real-time PCR were able to produce sensitive and accurate experimental results within hours instead of days, saving costs and reducing microbiology workloads.

No. of Stool Samples

181

par C positive excluded

178 3

MagNA Pure 96 / LightCycler PCR Routine microbiological testing Clinical Data

stx 1 positive

1

stx 2 positive

35

stx 1/2 positive

36

ESBL negative and Shiga Toxin 1 positive

1

ESBL positive and Shiga Toxin 2 positive

33

ESBL/EHEC negative and Shiga Toxin negative

2

34 cases of proven EHEC

infections including HUS

stx 1/2 negative

142

ESBL/EHEC negative and Shiga Toxin negative

142

No EHEC infection including HUS

in the follow up reported

together

corresponding to

corresponding to

Figure 3: Results of analytical data in correlation with clinical data.

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Conclusion

The workflow described here demonstrates that MagNA Pure 96 can efficiently extract DNA from EHEC in stool. In the 181 samples examined, the LightCyler® 480 confirmed, a sensitivity of 100% and a specificity of 98% in amplifying genes encoding Shiga toxins 1 and 2. In comparison to other screening assays which can take days, this automated

workflow produces high quality results within hours. Although further confirmatory tests will be required, the study described here using DNA extracted directly from stool specimens shows its applicability for fast screening in EHEC-urgent situations.

References

1. Reischl, U., M.T. Youssef, J. Kilwinski, N. Lehn, W.L. Zhang, H. Karch, and N.A. Strockbine (2002). Real-time fluorescence PCR assays for the detection and characterization of Shiga toxin, intimin and enterohemolysin genes from Shiga toxin-producing Escherichia coli.

J. Clin. Microbiol. 40:2555–2565. http://jcm.asm.org/cgi/content/short/40/7/2555

2. European Centre for Disease Prevention and Control and European Food Safety Authority. Shiga toxin/verotoxin-producing Escherichia coli in humans, food and animals in the EU/EEA, with special reference to the German outbreak strain STEC O104. Stockholm: ECDC; 2011

http://www.ecdc.europa.eu/en/publications/ Publications/1106_TER_EColi_joint_EFSA.pdf

3. Robert Koch-Institut. Informationen zum EHEC/HUS-Ausbruchsgeschehen. Stand 15.6.2011, 10.00 Uhr,

Datenstand 14.6.2011, 15.00 http://www.rki.de/cln_116/nn_205760/DE/Home/Info-

HUS.html

4. Kist M. et al. Infektionen des Darms. MiQ 9/2000 – Qualitätsstandards in der mikrobiologisch-infektio-logischen Diagnostik. Urban & Fischer Verlag

5. Konsiliarlabor für Hämolytisch-Urämisches Syndrom (HUS) am Institut für Hygiene · Universitätsklinikum Münster. Direktor: Prof. Dr. Dr. h.c. H. Karch: Laborinformationen zum EHEC Ausbruchsstamm (Stand 01.06.2011)

www.ehec.org/pdf/Laborinfo_01062011.pdf

Important Note: Regarding the specific assay(s) described, Roche was neither involved in establishing the experimental conditions nor in defining the criteria for the performance of the specific assays. Roche therefore cannot take any responsibility for performance or interpretation of results obtained for the biological target parameter(s) described by the authors or other users using a similar experimental approach. Potential users are informed to be aware of and in accordance with local regulations for assay validation and the scope of use for the involved Roche products, and to ensure that their use is valid in the countries where the experiments are performed. The LightCycler® 480 Real-Time PCR Instrument is for life science research only. Not for use in diagnostic procedures.

The MagNA Pure 96 Instrument (06 541 089 001) is for in vitro diagnostic use.

The LightCycler® 480 Instrument is for life science research only. Not for use in diagnostic procedures.

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