DETECTION OF HELICOBACTER PYLORI FROM FOOD SOURCES BY A NOVEL MULTIPLEX PCR ASSAY

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1 DETECTION OF HELICOBACTER PYLORI FROM FOOD SOURCES BY A NOVEL MULTIPLEX PCR ASSAY X. MENG 1,2, H. ZHANG 1,2,J.LAW 2, R. TSANG 2 and T. TSANG 1,2,3 1 NorthShore University HealthSystem 2650 Ridge Avenue Evanston, IL Northwestern University Feinberg School of Medicine 676 N. Saint Clair Street, #1400 Chicago, IL Accepted for Publication November 26, 2007 ABSTRACT Helicobacter pylori infection is worldwide and it has been recognized as an important cause of gastritis, gastric and duodenal ulcers. However, the modes of transmission of H. pylori infection are unclear. This study was designed to detect H. pylori from raw or ready-to-eat foods to provide more evidence for oral-oral and fecal-oral transmission patterns. For this purpose, a total of 11 fresh raw chickens from a local grocery and 18 orders of ready-to-eat raw tuna meat from a restaurant were collected. H. pylori were detected with our new multiplex polymerase chain reaction (PCR) assay, which can amplify 10 DNA fragments from five gene loci at the same time. H. pylori were positive in 36% (4/11) of the raw chickens and 44% (8/18) of the ready-to-eat raw tuna meat tested using the new multiplex PCR assay. Our results demonstrate that food might be a vehicle for H. pylori transmission among humans. PRACTICAL APPLICATIONS The prevalence of Helicobacter pylori infection may be as high as 80% in developing countries and up to 40% in developed countries. However, the 3 Corresponding author. T. Tsang, NorthShore University HealthSystem, Glenbrook Hospital, Room B067, 2100 Pfingsten Road, Glenview, IL 60026, U.S.A. TEL: ; FAX: ; doctat@aol.com Journal of Food Safety 28 (2008) All Rights Reserved. 2008, The Author(s) Journal compilation 2008, Wiley Periodicals, Inc. 609

2 610 X. MENG ET AL. mode of transmission, the natural history and other aspects of the epidemiology of H. pylori infection are still unclear. There have been no reports of H. pylori being cultured and isolated from foods. In this study we designed a one-step multiplex polymerase chain reaction assay to amplify 10 DNA fragments at the same time to detect H. pylori, which could overcome the difficulty in identifying H. pylori by using culture or other routine tests. Since this assay is so sensitive, specific and rapid, it has a great potential for identifying H. pylori, which is difficult to be cultured by using current methods, from different food samples. INTRODUCTION Helicobacter pylori is a source of a worldwide, highly prevalent, serious and chronic infection that has been linked causally with a diverse spectrum of gastrointestinal disorders including peptic ulcer disease, non-ulcer dyspepsia and gastric cancer (Blaser 1998; Moayyedi et al. 2003; Guarner 2004; De Luca and Iaquinto 2004). In early 1994, the International Agency for Research on Cancer classified H. pylori as a group 1 carcinogen. By country, the overall prevalence of H. pylori varies between 20 and 80% with the lowest overall prevalence of infection in North America and Western Europe and the highest prevalence in Eastern Europe, Asia and many developing countries (Kosunen et al. 1973; Cullen et al. 1993; Sonnenberg and Everhart 1996; Dunn et al. 1997). The route of H. pylori transfer is still speculative and controversial. Researchers have propounded oral-oral, fecal-oral and gastro-oral routes (Mapstone et al. 1993; Megraud 1995; Parsonnet et al. 1999). It is possible that all three routes may play a part in selected populations. H. pylori has been cultured from a small percentage of saliva and dental plaque samples examined (Li et al. 1995; Song et al. 2000a,b; Kabir 2004). Its presence in these samples indicates that the bacteria could be dispersed by an oral-oral route such as spitting, coughing or vomiting. The fact that H. pylori has also been isolated from feces supports the possible fecal-oral route (Thomas et al. 1992). H. pylori has been identified in different water sources (Hegarty et al. 1999; Engstrand 2001; Horiuchi et al. 2001; Rolle- Kampczyk et al. 2004). This suggests that water may be a vehicle for H. pylori transmission. The objective of this investigation was to detect H. pylori in ready-to-eat and raw food, in order to determine whether food is a vehicle for H. pylori transmission, and provide more evidence for oral-oral and fecal-oral transmission routes.

3 DETECTION OF H. PYLORI FROM FOOD SOURCES 611 MATERIALS AND METHODS Food Sample Eleven raw chicken samples (whole chicken with skin) were obtained from a grocery in the Chicago area. All chickens had been slaughtered within 24 h before transport to the grocery store and had been stored at 4C. Eighteen samples of raw tuna (Bluefin tuna; Thunnus thynnus) taken from ready-to-eat sushi at two restaurants in the Chicago area were also obtained for testing. Detection by Multiplex PCR All samples were washed thoroughly with PBS in stomacher bags and the solution was collected and then centrifuged at 2,000 rpm for 15 min (centrifuge: Beckman Allegra 21R, Beckman Coulter, Inc., Fullerton, CA). The supernate was further processed for DNA extraction and the DNA samples were ready for the detection of H. pylori. A novel multiplex polymerase chain reaction (PCR) method was developed to target the following loci: the urease gene (urea), 26 kda protein antigen, Hpa A gene, 0.86 kb DNA fragment and DNA sequences of 16S ribosomal RNA (Song et al. 1999; Li et al. 1995; Myung et al. 2000). For each locus, one forward primer was selected as the common primer (FC) and two reversed primers (R1, R2) were designed. R2 was located inside of the amplifying region of R1 and FC (all primers can be obtained by request). All five R1 primers and five R2 primers were mixed with five FC primers respectively, and set in two separate amplification systems. This would allow five DNA fragments to be amplified in each tube at the same time, and for each locus, two fragments, one internal to another, are expected to be amplified (Fig. 1). For each reaction, 2 ml of DNA template was added to 20 ml of a reaction mixture containing 10 mm Tris-HCl (ph 8.3), 50 mm KCl, 1.6 mm MgCl 2, 0.001% (w/v) gelatin, 0.3 mm of each deoxynucleotide and 0.05 mm of each oligonucleotide primer according to the two systems discussed previously. Immolase DNA polymerase (4.0 U; Bioline Ltd, London, U.K.) was used. The PCR was performed with a PCR thermal cycler (Marstercycler, Eppendorf Scientific Inc, Waterbury, NY) and the PCR conditions were as follows: an initial denaturation of target DNA at 94C for 5 min, followed by 41 cycles of 94C for 30 s, 56C for 50 s and 72C for 1 min. The final extension step was at 72C for 10 min. The PCR products were routinely analyzed on a 2.0% agarose gel by electrophoresis of a 10-mL aliquot and stained with ethidium bromide and visualized by excitation under UV light.

4 612 X. MENG ET AL. FIG. 1. DIAGRAM OF PRIMERS DESIGNED FOR EACH LOCUS. FC, THE FORWARD PRIMER IS THE COMMON PRIMER; R1 AND R2 ARE THE TWO REVERSED PRIMERS. AMPLICON FC-R1 AND FC-R2 ARE THE TWO DNA FRAGMENTS AMPLIFIED WITH FC-R1 AND FC-R2 FROM EACH LOCU, RESPECTIVELY RESULTS By using our novel multiplex PCR system, only 6 h were required to complete the detection. Thirty-six percent (4/11) of the fresh raw chickens and 44% (8/18) of the ready-to-eat raw tuna specimens were H. pylori-positive with the novel multiplex PCR testing method (Fig. 2). DISCUSSION In most studies one or two genes were used to identify H. pylori (Li et al. 1995; Myung et al. 2000). In our new test, 10 expected DNA fragments from the five selected loci in H. pylori DNA were obtained with our multiplex PCR detection system at the same time, and were displayed in a DNA ladder in the 2% agarose gel with ethidium bromide staining, including the 0.86 kb DNA fragment, 706 and 574 bp; Urea A gene, 526 and 465 bp; 16S rrna, 371 and 315 bp; 26 kda, 277 and 183 bp; Hpa A gene, 138 and 118 bp. In addition, the sensitivity of the one-step multiple-nested PCR assay was investigated. With 41 cycles of amplification, the one-step multiple-nested PCR was able to detect the 10 DNA bands in a single reaction when the DNA of H. pylori from the template was diluted to as little as pg (equal to about 300 single-copy DNA targets).

5 DETECTION OF H. PYLORI FROM FOOD SOURCES 613 FIG. 2. DETECTION OF H. PYLORI FROM FRESH RAW TUNA AND CHICKEN SAMPLES BY THE ONE-STEP MULTIPLEX SYSTEM (A) Tuna sushi samples: M: DNA marker. J99: standard H. pylori as positive control; S1, S2, and S4: H. pylori positive samples, For S1, S2, all 10 bands appeared; S3: H. pylori negative sample. (B) Fresh raw chicken samples. M: DNA marker. J99: standard H. pylori as positive control; C1 and C4: H. pylori positive samples; C2 and C3: H. pylori negative sample. To test the specificity of our multiplex PCR assay, 11 bacterial species were chosen, including: (1) Escherichia coli; (2) Enterobacter aerogenes; (3) E. cloacae; (4) Enterococcus species; (5) Viridians group, Streptococcus; (6) Pseudomonas aeruginosa; (7) Serratia species; (8) Klebsiella pneumoniae; (9) Methicillin resistant Staphylococcus aureus; (10) Lactobacillus species; and (11) Citrobacter species as templates. The PCR was performed in the conditions described along with H. pylori J99 as the positive control. None of those bacteria tested positive and showed the standard PCR band pattern as H. pylori. Comparing to conventional PCR, multiplex PCR method allowed for more accurate diagnosis of H. pylori. In this study, the amplified DNA fragments can be confirmed with our one-step multiplex PCR immediately because two fragments will be produced for each of the five loci, one internal to the other. The internal control of our one-step multiple-nested PCR is used

6 614 X. MENG ET AL. to rule out false positives caused by homological DNA sequences among various species in the primer binding sites. When both fragments are present for each locus, we can conclude with high confidence that the DNA sample is from H. pylori. Because of the high diversity of DNA sequences in H. pylori, in this study, we decided that the result of the one-step multiplex PCR assay is positive for H. pylori if 5/10 fragments or four DNA fragments from two loci are amplified. Because the PCR is so sensitive, it means that trace amounts of DNA contaminants could serve as templates, resulting in amplification of the wrong template (false positives). To avoid PCR contamination from sources such as: (1) laboratory benches, equipment and pipetting devices, which can be contaminated by previous DNA preparations; (2) cross-contamination between samples; and (3) products from previous PCR amplifications, we set up three physically separated working places for: template preparation before PCR; setting up PCR reactions; and post-pcr analysis. We also use special aerosolresistant pipette tips for our test and shining ultraviolet light on equipment, and pipetting devices to eliminate DNA contamination. Have a special set of PCR reagents and solutions that are used only for this study and store these reagents in small aliquots. To avoid H. pylori contamination from sample handling, once we got the test sample, we put them on a sterilized STOMACHER plastic bag and always used new sterilized plastic wares to prepare template DNAs. As a general rule, when setting up a PCR reaction, always include a negative ( no DNA ) control that contains all reaction components except the template DNA. To date, there is no convincing evidence that chicken, fish or any other animals are significant reservoirs of the H. pylori that is identified in human stomachs. Numerous workers have isolated H. pylori-like bacteria from monkeys (Shuto et al. 1993; Dubois et al. 1994); however, based on biochemical and phenotypic analysis, it is not proven that H. pylori isolated from those monkeys is highly related or identical to isolates from humans. Furthermore, considering the lack of close contact of the majority of humans with nonhuman primates, it is unlikely that nonhuman primate infection plays a significant role in causing human infection. Studies have demonstrated that H. pylori is capable of colonizing the stomachs of cats and pigs (O Rourke and Lee 2003; Koga et al. 2002). Contact with guinea pigs and sheep was shown to be a risk factor for H. pylori infection. However, there is no further evidence that these animals are natural reservoirs for H. pylori and sources of infection for humans. Thus, an animal-human route is not an accepted mode of transmission for H. pylori. Because it is highly unlikely that H. pylori resides in living chicken and tuna fish, we propose that the H. pylori detected in our samples of raw chicken from a grocery and in raw tuna from sushi restaurants were obtained when

7 DETECTION OF H. PYLORI FROM FOOD SOURCES 615 these foods were slaughtered and/or processed. Humans are the definitive natural reservoirs of H. pylori. Thus, it is likely that abattoir workers, chefs and other workers that come in close contact with foods may be the original source of the H. pylori found in our samples of chicken and tuna. The chicken and tuna fish might be contaminated through those workers hands, water, tools and equipment that they used. There was a study that tested the survival of H. pylori in ready-to-eat foods at 4C (Poms and Tatini 2001). The authors inoculated H. pylori in various semi-processed ready-to-eat foods and one raw chicken and observed the survival of H. pylori at 4C under aerobic conditions. H. pylori was recovered from spiked pasteurized milk and tofu samples up to 5 days later and from spiked leaf lettuce and raw chicken up to 2 days later. Our study builds upon this finding by demonstrating that H. pylori actually exist in some foods. Thus, it is highly probable that H. pylori could contaminate foods and survive in or on these foods for some time, being transmitted to those who consume it. This supports the propounded oral-oral and fecal-oral modes of transmission. However, there have been no reports of H. pylori being isolated and cultured from foods. This may be due to the fact that H. pylori are difficult to culture. When cultured in the laboratory, H. pylori usually grow slowly and do not compete well with other bacteria (Megraud 1997). This makes it difficult to isolate it from complex environmental samples. It may be that the available culture media and other conditions are not optimal for H. pylori growth in vitro. Researchers have found that H. pylori in adverse environments can transform into a viable but nonculturable state, a coccoid form (Bode et al. 1993; Chan et al. 1994). It has been speculated that the coccoid form of H. pylori plays a role in the survival of the bacterium outside the human host, which may contribute to the transmission of H. pylori from an environmental source. The coccoid form possesses the same enzymes and proteins that have been defined as virulence factors in spiral forms but with reduced enzyme and protein synthesis, and they also cause the same kind of inflammation and immune response in mice even when they are nonculturable (Wang et al. 1997). Coccoid forms of H. pylori seem to survive in stool samples and in water (Enroth et al. 1995; Lu et al. 2002). Therefore, coccoid forms of H. pylori may be important in food-borne transmission of the bacteria. The coccoid forms of H. pylori have not been culturable in most studies despite prolonged culture for up to 4 weeks (Cellini et al. 1994; Kuster et al. 1997). To overcome this problem, PCR-based methods have been used to detect H. pylori from environmental samples because the genome of H. pylori is still complete and viable after conversion to coccoid forms (Narikawa et al. 1997; Shahamat et al. 2004; Sisto et al. 2000; Zhang et al. 1999). Most previous PCR methods target one or more genes, and each PCR reaction is

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