A reverse transcriptase polymerase chain reaction assay for the detection of thermophilicCampylobacterspp.

Molecular and Cellular Probes (1998) 12, 317–322 Article No. ll980184

A reverse transcriptase polymerase chain reaction assay for the detection of thermophilic Campylobacter spp. A. D. Sails,1∗ F. J. Bolton,1 A. J. Fox,2 D. R. A. Wareing1 and D. L. A. Greenway3 1

Preston Public Health Laboratory, The Royal Preston Hospital, PO Box 202, Sharoe Green Lane, Fulwood, Preston, Lancashire, PR2 9HG, UK, 2Manchester Public Health Laboratory, Withington Hospital, Nell Lane, West Didsbury, Manchester, M20 2LR, UK and 3The Department of Applied Biology, The University of Central Lancashire, Preston, Lancashire, PR1 2HE, UK (Received 4 March 1998, Accepted 16 June 1998) A novel method was developed for the detection of thermophilic enteropathogenic campylobacters based on the detection of mRNA using the reverse transcriptase polymerase chain reaction (RTPCR). The RNA extraction method, DNase treatment and RT-PCR assay were shown to be specific for mRNA. The assay is specific for the thermophilic campylobacters Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis and restriction fragment length polymorphism (RFLP) analysis of the 256 bp amplified product with the restriction endonucleases Alu I, Dde I and Dra I revealed distinct species specific patterns. The assay was applied to the detection of C. jejuni cells killed by heating at 72°C for 5 min and mRNA was detected by RT-PCR immediately after heat killing but became undetectable within 4 h when the cells were held at 37°C. The assay therefore can differentiate between viable and dead cells of C. jejuni.  1998 Academic Press

KEYWORDS: Campylobacter, reverse transcriptase, polymerase chain reaction, viability, mRNA.

INTRODUCTION

Campylobacter jejuni and Campylobacter coli are the most common cause of bacterial gastro-enteritis in humans.1 Epidemiological evidence suggests that animals, particularly poultry, cattle, wild birds, pigs and domestic pets are reservoirs for the strains infecting humans.2,3 Untreated milk4 and untreated water5 have been associated with large outbreaks of Campylobacter gastro-enteritis; however, the majority of outbreaks are not confirmed microbiologically.6 More effective methods for the detection of campylobacters may facilitate microbiological confirmation of infection. The majority of Campylobacter infections appear to be sporadic and in most cases the sources

of infection are undetermined.7 It is therefore important to clarify the role of natural reservoirs, animals and food in the transmission of Campylobacter infection. A rapid, sensitive and specific method for detecting viable enteropathogenic campylobacters in complex substrates such as foods, dairy products and environmental samples is essential to further understanding of the epidemiology of infection and to enable the tracking of C. jejuni through the foodchain. It would also facilitate the design and implementation of effective intervention measures. Traditional methods for the isolation of campylobacters

∗ Author to whom all correspondence should be addressed at: Preston Public Health Laboratory, The Royal Preston Hospital, PO Box 202, Sharoe Green Lane, Fulwood, Preston, Lancashire PR2 9HG, UK.

0890–8508/98/050317+06 $30.00/0

 1998 Academic Press

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from foods and related environments are slow and can require incubation periods in excess of 92 h.8 Furthermore, the phenotypic methods of identification and speciation require complex biochemical tests which can be subjective in their interpretation and may lead to misidentification.9 Recent work has demonstrated that environmentally stressed campylobacters may persist in the environment in a ‘nonculturable viable’ (NCV) form which can be recovered in vivo.10 The polymerase chain reaction (PCR) is a highly sensitive, specific and rapid method for detecting bacteria in pure cultures and natural reservoirs.11 There have been several reports of using PCR methods for the detection of C. jejuni in water,11,12 chicken13,14 and dairy products.15–17 The primers of Jackson and colleagues produce an amplicon which when digested with the restriction enzymes Alu I, Dde I and Dra I yield species specific restriction fragment length polymorphism (RFLP) patterns. However, these conventional PCR methods detect chromosomal gene sequences which can be present in non-viable cells and therefore cannot determine viability.18 Detection of viable cells using messenger RNA (mRNA) as the target for reverse transcriptase PCR (RT-PCR) has been demonstrated in Legionella pneumophila,19 Mycobacterium leprae,20 Mycobacterium tuberculosis 21 and Listeria monocytogenes.22 The half-life of some bacterial mRNA species has been demonstrated to be less than 2 min23 therefore dead cells should contain no mRNA. The objective of this study was to develop an RT-PCR assay for the detection of mRNA from thermophilic campylobacters based upon the primers of Jackson and colleagues17 and apply it to the detection of viable and dead cells of C. jejuni. MATERIALS AND METHODS Bacterial strains and growth conditions The Campylobacter species used in this study were C. jejuni (NCTC 11168), C. coli (NCTC 11366) and C. upsaliensis (NCTC 11540). Prior to nucleic acid extraction the Campylobacter strains were grown overnight on charcoal, ferrous sulphate, sodium pyruvate (CFP) blood-free medium24 at 37°C for 24 h. All cultures were incubated in micro-aerobic conditions, obtained using the evacuation replacement technique.25 RNA extraction Extraction of total RNA from bacterial strains was performed using a Micro-Scale Total RNA separation

Table 1. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR results of the DNase and RNase treatment of RNA extracted from Campylobacter jejuni (NCTC-11168) PCR resulta

Enzymatic treatment of RNA extract

DNase

<30 U 30 U >40 U

RNase DNase (30 U) and RNase a

RT-PCR (mRNA)

PCR

+ + ± + −

± − − + −

+, DNA or RNA amplified; −, no amplification.

kit (Clontech Laboratories Inc., USA). Bacterial cells were harvested from fresh overnight plate cultures and a suspension of 1×108 cells was prepared in denaturing solution. Total cellular RNA was extracted according to the manufacturer’s protocol. The RNA extract obtained was resuspended in 40 ll Di-ethyl pyrocarbonate (DEPC) treated sterile water (Fluka Biochem, UK). Chromosomal DNA was eliminated from a 20 ll aliquot of the RNA extract by adding 30 U DNase, RNase-free (Boehringer Mannheim, UK) in the presence of 21 U RNAguard Ribonuclease inhibitor (Pharmacia Biotech, UK). The reaction mixture was incubated at 37°C for 1 h, and the DNase enzyme subsequently inactivated by heating the reaction mixture at 90°C for 5 min.

Reverse transcriptase polymerase chain reaction Reverse transcriptase polymerase chain reaction was performed on the RNA extract using the EZ rTth RNA PCR kit (PE-Applied Biosystems, UK). The recombinant Thermus thermophilus (rTth) polymerase used in this system is a thermoactive RNA polymerase and a thermostable DNA polymerase. Reverse transcriptase polymerase chain reaction was performed in a 50 ll volume reaction mix containing: 19·5 ll DEPC-treated sterile water, 10·0 ll 5×EZ buffer (250 m Bicine, 575 m potassium acetate, 40% {w/v} glycerol, pH 8·2), 6 ll dNTP mix (final concentration 300 l), 2·0 ll rTth DNA polymerase (final concentration 5 units), 5 ll 25 m manganese acetate (final concentration 2·5 m), 1·25 ll primers 1 and 217 (final concentration 0·5 l), prepared in thin walled 0·5 ml PCR microtubes and overlaid with 50 ll mineral oil (Sigma, UK). Five microlitres of DNase treated RNA template were added through the mineral oil overlay.

Campylobacter RT-PCR 1

2

3

4

5

6

7

8

319 9

10

11

12

300 200 100

Fig. 1. Restriction fragment length polymorphism (RFLP) patterns for three Campylobacter species. Lanes 1, 12: 100 bp ladder (100, 200, 300 bp etc.); 2: unrestricted 256 bp Campylobacter jejuni amplimer (control); 3: C. jejuni Alu I digest (108, 148); 4: Campylobacter coli Alu I digest (108, 148); 5: Campylobacter upsaliensis Alu I digest (256); 6: C. jejuni Dde I digest (83, 173); 7: C. coli Dde I digest (83, 173); 8: C. upsaliensis Dde I digest (30, 83, 143); 9: C. jejuni Dra I digest (123, 133); 10: C. coli Dra I digest (256); 11: C. upsaliensis Dra I digest (256).

The RT-PCR cycle was 60°C for 30 min (reverse transcription); 95°C for 2 min; and 40 cycles of 94°C for 25 s, 55°C for 40 s, 72°C for 60 s; and a terminal extension step of 72°C for 5 min. Polymerase chain reaction products were analysed by gel electrophoresis using ethidium bromide staining.

Dde I and Dra I according to the manufacturer’s instructions. Fragments were separated using agarose gel electrophoresis and visualized by ethidium bromide staining.

Detection of heat-killed C. jejuni cells by RTPCR and conventional PCR Verification of RT-PCR To confirm that the RT-PCR amplification product of the assay was derived from RNA and not contaminating chromosomal DNA, total RNA was extracted from C. jejuni NCTC-11168. Aliquots of the RNA were mixed with 30 U DNase, RNase-free to eliminate any DNA present in the extracts or 1·0 lg RNase, DNase-free (Boehringer Mannheim, UK) to eliminate RNA from the extracts. A number of aliquots were treated with both DNase and RNase to eliminate both DNA and RNA from the extracts. Aliquots were then used as template in both RT-PCR and conventional PCR (without RT).

Identification of Campylobacter species using RFLP analysis of PCR products Aliquots (10 ll) of the amplification products were digested with the restriction endonucleases Alu I,

Campylobacter jejuni (NCTC-11168) was grown overnight in 100 ml Campylobacter enrichment broth (including growth supplement; Lab M, UK) at 42°C to obtain a cell density of 108 cfu ml−1 (confirmed by Miles and Misra surface counts). Aliquots of the incubated broth (0·5 ml) were transferred into 0·7 ml microcentrifuge tubes. These were either maintained at 37°C by immersing in a water bath (non-heattreated control suspensions) or were subjected to heat treatment by immersion in a water bath maintained at 72°C for 5 min. Following heat-killing cell suspensions were either immediately harvested by centrifugation at 14 000 rpm (4°C) for 10 min or maintained at 37°C and harvested by centrifugation at 30 min, 1 h, 2 h and 4 h post heat-killing. Aliquots (0·5 ml) of heat-treated cells were also used to inoculate 100 ml of Campylobacter enrichment broth (containing 5% lysed horse blood) which was incubated at 37°C overnight and then at 42°C for 72 h in total. The broths were subcultured onto Columbia

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Table 2. Reverse transcriptase polymerase chain reaction (RT-PCR) and conventional PCR results of heattreated cells of Campylobacter jejuni (NCTC-11168) Untreated cells

Heat-treated cellsa 0b 0·5 1·0 2·0 4·0

RT-PCR Conventional PCR

+

+

+

+

+

−

+

+

+

+

+

+

+, DNA or RNA amplified. −, No amplification. a 72°C for 5 min. b Time of assay after heat treatment (h).

Blood agar plates (Oxoid Ltd, UK) containing 5% whole horse blood and incubated micro-aerobically at 37°C for 48 h. Total RNA was isolated from harvested cells as outlined above and treated with 30 U DNase. The RNA was then used as template in both RT-PCR and conventional PCR (without RT). DNA was also isolated from harvested cell suspensions using the Isoquick Total Nucleic Acid Extraction kit (Orca Research Inc, USA) according to the manufacturer’s instructions and the DNA was used as template in conventional PCR.

RESULTS AND DISCUSSION The recombinant Thermus thermophilus (rTth) DNA polymerase used in the assay has the ability to both reverse transcribe RNA and amplify DNA, therefore any DNA present in the RNA extract can lead to false positive results. To verify that the product in the RTPCR assay was derived from RNA and not contaminating chromosomal DNA, total RNA from C. jejuni (NCTC-11168) was isolated and subjected to either DNase, RNase, or DNase and RNase treatments (Table 1). The RT-PCR assay was positive following DNase treatment of the template indicating that the amplified product originated from messenger RNA. Furthermore, conventional PCR was negative, confirming that the DNA had been successfully eliminated from the RNA extract. The RT-PCR was positive with RNase treatment, indicating that contaminating chromosomal DNA was present in the untreated RNA extract which was amplified by the rTth DNA polymerase enzyme. Additionally, conventional PCR on RNase treated template was positive confirming these results. The RT-PCR was negative following DNase and RNase treatment of the extract and therefore all template RNA and DNA had been eliminated. Previous studies have demonstrated that 3 U DNase are sufficient to eliminate DNA from RNA extracts of

Pseudomonas putida26 and 20 U DNase are sufficient to eliminate DNA from RNA extracts of Mycobacterium tuberculosis 21 prior to RT-PCR. However, initial studies conducted during the development of this assay demonstrated that 10 U DNase did not effectively eliminate contaminating chromosomal DNA from the campylobacter RNA preparations, thus leading to false positive results. A range of DNase concentrations of 10–100 U was investigated (data not shown) to determine the optimum amount of DNase to eliminate DNA from RNA preparations from campylobacter, prior to RT-PCR. Levels of DNase below 30 U were ineffective in eliminating contaminating chromosomal DNA from RNA preparations and DNase levels of greater than 40 U had inhibitory effects on the RT-PCR assay leading to a reduction in sensitivity (Table 1). Therefore optimization of DNase treatment of RNA preparations prior to RT-PCR is an essential prerequisite for definitive evidence of mRNA amplification in bacteria and the avoidance of false positive results. The primers of Jackson and colleagues17 amplify a 256 bp product from the sequence of an open-reading frame adjacent to and downstream of a novel twocomponent regulatory gene and were originally developed for use in a conventional PCR assay for the detection of thermophilic campylobacters. These primers were demonstrated to produce a 256 bp amplicon from C. jejuni, C. coli and C. upsaliensis which were shown to have species specific motifs. Digestion of the product with the restriction enzymes Alu I, Dde I and Dra I produces distinctive RFLP patterns specific for the three species.17 A RT-PCR assay for thermophilic campylobacters has been developed using these primers which also produces a 256 bp amplification product from C. jejuni, C. coli and C. upsaliensis. Digestion of the products from the RT-PCR assay with the restriction enzymes Alu I, Dde I and Dra I yielded RFLP patterns for C. jejuni, C. coli and C. upsaliensis that were identical to those originally reported by Jackson and colleagues (Fig. 1). These findings require confirmation using further isolates of these species. Additionally, the specificity of the assay should be investigated with a diverse range of other Campylobacter species and related organisms. In this study the authors have developed a RT-PCR assay specific for mRNA from thermophilic campylobacters. The detection of mRNA in bacterial cells by RT-PCR has previously been demonstrated to be associated with viability, therefore the assay was applied to the detection of C. jejuni cells which had been killed by heating at 72°C to determine if the assay could differentiate between viable and nonviable cells. RNA was extracted from heat-treated

Campylobacter RT-PCR

suspensions of cells and used as template in both RTPCR and conventional PCR (Table 2). Resuscitation of the heat-treated cells using extended enrichment culture was negative, indicating the cells were not recoverable using current culture methods. Conventional PCR using the RNA extracts as template was negative, indicating that the DNase step eliminated DNA from the extracts. Reverse transcriptase polymerase chain reaction demonstrated that immediately after heat-treatment specific mRNA was still present in the cells at detectable levels. Furthermore mRNA could still be detected up to 3 h post heattreatment. Conversely, conventional PCR (without RT) demonstrated that DNA was present at detectable levels for greater than 24 h (data not shown). Therefore the assay has the ability to differentiate between viable and dead cells; however, the time the assay is applied to heat-killed cells is critical to ensure that residual mRNA is totally degraded by cellular nucleases. The effect of killing Escherichia coli cells by heat or ethanol prior to RT-PCR of mRNA from three gene targets was recently reported.27 mRNA was detected from all three genes immediately the cells had been killed by ethanol or heat but gradually disappeared with time. In the heat-killed cells the mRNA targets became negative after 2–16 h; however, after ethanol treatment mRNA was still detectable after 16 h. These differences must be related to the different effects of heat or ethanol treatment on the capacity for RNA breakdown in the dead cells. There have been no reports of the factors which are important in the breakdown of mRNA in dead cells, although it is thought to involve the action of cellular nucleases (RNases). Therefore if the treatment used to kill the cells has no effect on the cellular RNases then mRNA would be more rapidly degraded than in cells which had been killed using a treatment which inactivated the cellular RNases or protected the mRNA transcripts from attack. Methods of killing bacterial cells must be investigated further to establish the factors which influence the rate of mRNA degradation in dead Campylobacter cells and the effects of the post-treatment holding conditions. This will facilitate application of the assay to the detection of viable cells in foodstuffs, clinical and environmental samples. Additionally, this assay may be able to determine the viability of non-culturable viable Campylobacter cells. ACKNOWLEDGEMENTS This work was supported by The Ministry of Agriculture and Fisheries and Foods research programme number FS1242.

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REFERENCES 1. Butzler, J. P., Glupcynski, Y. & Goossens, H. (1992). Campylobacter and Helicobacter Infections. Current Opinions in Infectious Diseases 5, 80–7. 2. Blaser, M. J., Taylor, D. N. & Feldman, R. A. (1983). Epidemiology of Campylobacter jejuni infections. Epidemiology Reviews 5, 157–76. 3. Jones, D. M., Abbott, J. D., Painter, M. J. & Sutcliffe, E. M. (1984). Comparison of biotypes and serotypes of Campylobacter spp. isolated from patients with enteritis and from animal sources. Journal of Infection 9, 51–8. 4. Vogt, R. L., Little, A. A., Patton, C. M., Barrett, T. J. & Oriari, L. A. (1984). Serotyping and serology studies of Campylobacteriosis associated with consumption of raw milk. Journal of Clinical Microbiology 20, 998–1000. 5. Penner, J. L., Pearson, A. D. & Hennessey, J. N. (1983). Investigation of a waterborne outbreak of Campylobacter jejuni enteritis with a serotyping scheme based on thermostable antigens. Journal of Clinical Microbiology 18, 1362–5. 6. Pebody, R. G., Ryan, M. J. & Wall, P. G. (1997). Outbreaks of campylobacter infection: rare events for a common pathogen. CDR Review 7, 33–7. 7. Cowden, J. (1992). Campylobacter: epidemiological paradoxes. British Medical Journal 305, 132–3. 8. Corry, J. E. L., Post, D. E., Colin, P. & Lasiney, M. J. (1995). Culture media for the isolation of campylobacter. International Journal of Food Microbiology 26, 43–76. 9. On, S. L. & Holmes, B. (1992). Assessment of enzyme detection tests useful in identification of campylobacters. Journal of Clinical Microbiology 30, 746–9. 10. Jones, D. M., Sutcliffe, E. M. & Curry, A. (1991). Recovery of viable but non-culturable Campylobacter jejuni. Journal of General Microbiology 137, 2477–82. 11. Bej, A. K., Steffan, R. J., DiCesare, J., Haff, L. & Atlas, R. M. (1990). Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Applied and Environmental Microbiology 56, 307–14. 12. Hernandez, J., Alonso, J. L., Fayos, A., Amoros, I. & Owen, R. J. (1995). Development of a PCR assay combined with a short enrichment culture for the detection of Campylobacter jejuni in estuarine surface waters. FEMS Microbiology Letters 127, 201–6. 13. Giesendorf, B. A. J., Quint, W. G. V., Henkens, M. H. C., Stegeman, H., Huff, F. A. & Niesters, H. G. M. (1992). Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3804–8. 14. Itoh, R., Saitoh, S. & Yatsuyanagi, J. (1995). Specific detection of Campylobacter jejuni by means of the polymerase chain reaction in chicken litter. Journal of Veterinary Medicine & Science 57, 125–7. 15. Wegmuller, B., Luthy, J. & Candrian, U. (1993). Direct polymerase chain reaction of Campylobacter jejuni and Campylobacter coli in raw milk and dairy products. Applied and Environmental Microbiology 59, 2161–5. 16. Allmann, M., Hofelein, C., Koppel, E., Luthy, J., Meyer, R., Niederhauser, C., Wegmuller, B. & Candrian, U. (1995). Polymerase chain reaction (PCR) for detection

322

17.

18.

19.

20.

21.

A. D. Sails et al. of pathogenic microorganisms in bacteriological monitoring of dairy products. Research in Microbiology 146, 85–97. Jackson, C. J., Fox, A. J. & Jones, D. (1996). A novel polymerase chain reaction assay for the detection and speciation of thermophilic Campylobacter spp. Journal of Applied Bacteriology 81, 467–73. Josephson, K. L., Gerba, C. P. & Pepper, I. L. (1993). Polymerase chain reaction detection of nonviable bacterial pathogens. Applied and Environmental Microbiology 59, 3513–5. Bej, A. K., Mahbubani, M. H. & Atlas, R. M. (1991). Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods. Applied and Environmental Microbiology 57, 597–600. Patel, B. K., Banerjee, D. K. & Butcher, P. D. (1993). Determination of Mycobacterium leprae viability by polymerase chain reaction amplification of 71 kDa heat-shock protein mRNA. Journal of Infectious Disease 168, 799–800. Jou, N.-T., Yoshimori, R. B., Mason, G. R., Louie, J. S. & Liebling, M. R. (1997). Single-Tube, nested, reverse transcriptase PCR for detection of viable Mycobacterium tuberculosis. Journal of Clinical Microbiology 35, 1161–5.

22. Herman, L. (1997). Detection of viable and dead Listeria monocytogenes by PCR. Food Microbiology 14, 103–10. 23. Belasco, J. G. & Higgins, C. F. (1988). Mechanisms of mRNA decay in bacteria: a perspective. Gene 72, 15–23. 24. Bolton, F. J., Holt, A. V. & Hutchinson, D. N. (1984). Campylobacter biotyping scheme of epidemiological value. Journal of Clinical Pathology 37, 677–81. 25. Bolton, F. J., Wareing, D. R. A., Skirrow, M. B. & Hutchinson, D. N. (1992). Identification and Biotyping of Campylobacters. In Identification Methods in Applied and Environmental Microbiology (Board, R. G., Jones, D. & Skinner, F. A., eds) pp. 151–62. Oxford, England: Blackwell Scientific Publishers. 26. Selvaratnam, S., Schoedel, B. A., McFarland, B. L. & Kulpa, C. F. (1995). Application of reverse transcriptase PCR for monitoring expression of catabolic dmpN gene in a phenol-degrading sequencing batch reactor. Applied and Environmental Microbiology 61, 3981–5. 27. Sheridan, G. E. C., Masters, C. I., Shallcross, J. A. & Mackey, B. M. (1998). Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli cells. Applied and Environmental Microbiology 64, 1313–18.