VacA, cagA, iceA and oipA genotypes status and antimicrobial resistance properties of Helicobacter pylori isolated from various types of ready to eat foods

Despite the high clinical standing of Helicobacter pylori, its exact routes of transmission and origin have not been determined. Based on the contentious hypothesis, foods play an important roles in the transmission of H. pylori to humans. The present study was carried out to investigate the vacA, cagA, oipA and iceA genotypes status of H. pylori isolated from the various types of ready to eat foods. A total of 550 ready to eat food samples were cultured and tested. H. pylori-positive strains were analyzed for the presence of various genotypes and antimicrobial resistance pattern. Seventy four out of 550 (13.45 %) samples were positive for H. pylori. Olvie salad (36 %), restaurant salad (30 %), fruit salad (28 %) and soup (22 %) were the most commonly contaminated. H. pylori strains harbored the highest levels of resistance against amoxicillin (94.59 %), ampicillin (93.24 %), metronidazole (89.18 %) and tetracycline (72.97 %). The most commonly detected genotypes were vacA s1a (78.37 %), vacA m2 (75.67 %), vacA m1a (51.35 %) and cagA (41.89 %). The prevalence of iceA1, iceA2 and oipA genotypes were 13.51, 4.05 and 18.91 %, respectively. S1am2 (70.27 %), s1am1a (39.18 %) and m1am2 (31.08 %) were the most commonly detected combined genotypes. Of 40 different genotypic combinations, s1a/cagA+/iceA1/oipA− (12.16 %), s1a/cagA+/iceA1/oipA+ (10.81 %) and s1a/cagA−/iceA1/oipA+ (10.81 %) were the most prevalent. The present investigation showed that some types of ready to eat food samples maybe the sources of resistant and virulent strains of H. pylori. Warily use of antibiotics with respect to the results of disk diffusion method and careful health monitoring on food and staffs of food producing companies maybe reduce the risk of H. pylori in foods.


Background
Ready to eat foods play an important roles in the nutrition of Iranian people. Every day millions of people use from ready to eat foods in their main meals. Therefore, hygienic quality of ready to eat foods is extremely important regarding public health hazards.
Helicobacter pylori (H. pylori) is a microaerophilic gram-negative bacterium which is known as the causative agent of peptic ulcer disease, type B gastritis, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma [1]. It has been estimated that 17-86 % of hospitalized patients with peptic ulcers were infected with H. pylori [1][2][3].
The role of foods in the transmission of H. pylori is still unknown but there were several investigations which focused on the identification of this bacterium in various types of food samples [4][5][6][7][8]. Good conditions for the growth of bacteria in various types of foods cause it to be survive [6,8].
To appraise the pathogenicity of H. pylori, evaluation of latent virulence factors and genotypes is essential. The most commonly important virulence factors among H. pylori strains of different clinical outcomes of human and animal beings are the vacuolating cytotoxin (vacA), induced by contact with the epithelium antigen (iceA), cytotoxin associated gene (cag) and outer inflammatory protein (oip) [6,9,10]. These genes are usually induced adhesion and invasion to the gastric epithelial cells [11,12]. The vacA belongs to the group of genes with variable genotypes or structures. This gene is associated with injury to epithelial cells. The vacA gene is polymorphic, comprising variable signal regions (type s1 or s2) and mid-regions (type m1 or m2). The s1 type is further subtyped into s1a, s1b and s1c subtypes and the m1 into m1a and m1b subtypes. The mosaic combination of s and m-region allelic types determines the particular cytotoxin and consequently, the pathogenicity of the bacterium [11,13]. The iceA gene has two main allelic variants iceA1 and iceA2 but their functions are not yet clear. Cag pathogenicity island (PAI) has been shown to be involved in persuading inflammation, ulceration and carcinogenesis [12]. The cagA gene has been detected in the specimens taken from the severe cases of peptic ulcer [8][9][10][11][12]. The oipA gene plays a significant role in effective colonization of mucosa [8][9][10][11][12]. Genotyping using these virulence markers is considered as one of the best approaches for study of correlations between H. pylori isolates from different samples.
Data on the epidemiology and transmission of H. pylori is extremely significant in order to prevent its distribution and to identify high-risk populations, especially in areas that have high rates of gastritis, peptic ulcers and gastric cancer such as Iran [6][7][8][9][10]13]. Considering the unclear epidemiological aspects of H. pylori in ready to eat foods and according to the high prevalence of H. pylori all-around the world, the present investigation was carried out in order to study the exact status of vacA, cagA, iceA and oipA genotypes and antibiotic resistance pattern of H. pylori isolated from various types of ready to eat food samples.

DNA extraction and Helicobacter pylori 16S rRNA gene amplification
Suspected colonies were identified as H. pylori based on the PCR technique. Genomic DNA was extracted from the colonies with typical characters of H. pylori using a DNA extraction kit for cells and tissues (Roche Applied Science, Germany, 11814770001) according to the manufacturer's instructions and its density was assessed by optic densitometry. Extracted DNA was amplified for the 16S rRNA gene (primers: HP-F: 5′-CTGGAGAGACTAAGCCCTCC-3′ and HP-R: 5′-ATTACTGACGCTGATTGTGC-3′) [14]. PCR reactions were performed in a final volume of 50 µL containing 5 µL 10 × buffer + MgCl 2 , 2 mM dNTP, 2 unit Taq DNA polymerase, 100 ng genomic DNA as a template, and 25 pmol of each primer. PCR was performed using a thermal cycler (Eppendorf Co., Germany) under the following conditions: an initial denaturation for 2 min at 94 °C; 30 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 8 min.
The PCR were performed in a total volume of 50 μl containing 1 μM of each primers, 1 μL of genomic DNA (approximately 200 ng), 1 mM of dNTPs mix (invitrogen), 2 mM of Mgcl2, and 0.05 U/μL Taq DNA polymerase (invitrogen). PCR amplifications were performed in an automated thermal cycler (Biometra Co., Germany). The following cycle conditions were used for PCR amplification: for vacA: 32 cycles of 45 s at 95 °C, 50 s at 64 °C, and 70 s at 72 °C; for cagA: 1 min at 94 °C, 1 min at 56 °C, and 1 min at 72 °C; for iceA: 1 min at 94 °C, 1 min at 56 °C, and 1 min at 72 °C and finally, for oipA: 1 min at 94 °C, 1 min at 56 °C and 1 min at 72 °C. All runs included one negative DNA control consisting of PCR grade water and two or more positive controls (26695, J99, SS1, Tx30, 88-23 and 84-183).

Gel electrophoresis
The PCR amplification products (10 μl) were subjected to electrophoresis in a 1.5 % agarose gel in 1X TBE buffer at 80 V for 30 min, stained with ethidium bromide, and images were obtained in a UVIdoc gel documentation systems (UK). The PCR products were identified by 100 bp DNA size marker (Fermentas, Germany).

Statistical analysis
Data were transferred to Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA, USA) for analysis. Using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA), Chi square test and Fisher's exact two-tailed test analysis was performed and differences were considered significant at values of P < 0.05. Distribution of H. pylori genotypes isolated from food stuffs were statistically analyzed.

Prevalence of Helicobacter pylori in various types of ready to eat food samples
All of the ready to eat food samples were examined using the culture and PCR techniques. Table 2 shows   (22 %). There were no positive results for sausage, salami and chicken nugget. There were statistically significant differences amongst the incidence of bacteria in hamburger and olvie salad (P = 0.027), traditional bread and restaurant salad (P = 0.033) and soup and falafel (P = 0.041).

Antimicrobial susceptibility pattern
Antimicrobial susceptibility of H. pylori isolates of ready to eat food samples is shown in Table 3.

Discussion
The results of our study showed that 13.45 % of ready to eat food samples were contaminated with H. pylori which was entirely high. In comparison with other investigations which were conducted on food staffs, the prevalence of H. pylori in our study was higher than those of milk (12.5 %) [22] and vegetable and salad (10.86 %) [7] but was lower than dairy products (19.2 %) [6], vegetable (13.68 %) [8] and restaurant salad (14 %) [8]. To date, various studies have been conducted on the prevalence of H. pylori in foods with animal origin [22][23][24][25]. In a study accompanied in Italy [23], the H. pylori was detected in more than 25 % of foods with animal origin. Japanese researchers reported the higher levels of contamination in foods with animal origin (72.2 %) [26]. In despite of other foods with animal origin [22][23][24][25][26], prevalence of H. pylori in the hamburger, olvie salad and soup (meat based ready to eat food stuffs) of our study were 2, 36 and 22 %, respectively. Prevalence of bacterium in Greek [24] and USA [25] were 20 % and 60 %, respectively which was higher than our results. One of the most important substances in the preparation of sausage, salami, hamburger, olvie salad, chicken nugget and soup is meat. There were no positive results for chicken nugget, sausage and salami samples of our study. It is maybe related to the application of high temperature during processing of these products. In addition, observation of hygienic conditions in the preparation and packaging of these products maybe another reason for lack of H. pylori. In comparison with sausage, salami and chicken nugget samples, the H. pylori had significant prevalence in hamburger (2 %), olvie salad (36 %) and soup (22 %) samples. High amount of water activity (Aw) in these food samples, optimum pH and salt levels, presence of appropriate levels of amino acids including arginine, histidine, isoleucine, leucine, methionine, phenylalanine, alanine, valine, proline, serine, and tryptophan which are necessary for growth of H. pylori [27], presence of vegetables in some of these foods (olvie salad and soup) which are maybe reservoir for H. pylori [7,8], unsanitary conditions in their preparation and finally Table 3 Antimicrobial resistance pattern of Helicobacter pylori isolates of ready to eat food samples Cream-candy (9 cross contamination of these foods due to handling by factory and food units staffs are the main reasons for the high prevalence of H. pylori in these samples. The results of Mhaskar et al. [28] confirmed the finding of our study about the risk of meat products for H. pylori infection. They reported that the prevalence of peptic ulcer and H. pylori infection were entirely higher in those patients which were used from meat and meat products [odds ratio (OR) 2.35, 95 % confident interval (CI): 1. 30-4.23] and restaurant foods (OR 3.77, 95 % CI 1.39-10.23) than vegans.
Preparation of some kinds of these food samples including falafel (pea based fast food with some kinds of vegetables), cream-candy, restaurant salad, traditional bread, fruit salad and olvie salad need moderate levels of water. Roles of contaminated water and even drinking water in transmission of H. pylori have been confirmed previously [29][30][31][32]. Probably, the water sources used for production and processing of these foods samples were contaminated. Finally, using from unsanitary conditions, handling contamination, use of contaminated equipment and lack of public and individual hygiene are the main reasons for the high prevalence of H. pylori in food samples of our study. Food safety regulations as well as quality standards-including good agricultural practices (GAPs), good manufacturing practices (GMPs) and hazard analysis and critical control points (HACCP)-should be introduced in Iranian food units and factories in order to control contamination and proliferation of pathogenic bacteria.
Another part of our investigation focused on the distribution of vacA, cagA, oipA and iceA genotypes. Results showed that vacA s1a (78.37 %), vacA m2 (75.67 %), vacA m1a (51.35 %) and cagA (41.89 %) were the most commonly detected genotypes in H. pylori strains of ready to eat foods. Our results showed that the cagA gene had the highest prevalence in the hamburger (100 %), soup (81.81 %) and olvie salad (83.33 %). As we said, these are a meat based ready to eat food samples and the high prevalence of cagA gene in these samples maybe showed the high presence of cagA positive strains of H. pylori in the meat used for production of these food stuffs. Yahaghi et al. [8] reported that the oipA (86.44 %), cagA (57.62 %), vacA s1a (37.28 %) and vacA m1a (30.50 %) were the most commonly detected genotypes in the H. pylori isolates of vegetable and salad. They showed that vacA s1c   [9,10,13,33], foods [6,8]) and those of animal sources [9]. Close association of vacA and cagA genotypes with interleukin 8 (IL-8) production, cytotoxin production, gastric epithelial cells adhesion, inflammatory effects, vacuolization and apoptosis in gastric epithelial cells has been observed previously [34,35]. Since H. pylori isolates in our study harbored vacA and cagA genotypes, consumption of ready to eat foods contaminated with virulent strains may provoke duodenal ulceration, gastric mucosal atrophy and gastric cancer. Another important finding of our investigation is about the presence of high antibiotic resistance in the H. pylori strains of ready to eat food samples. Our results revealed that the H. pylori strains of ready to eat food samples had the high levels of antibiotic resistance against ampicillin, metronidazole, erythromycin, amoxicillin, tetracycline and trimethoprim. Similar findings have been reported previously by Yahaghi et al. [8], Thyagarajan et al. [36], Secka et al. [37] and Bang et al. [38]. Low levels of antibiotic resistance in the H. pylori strains against spiramycin, furazolidone, cefsulodin, rifampin, streptomycin and levofloxacin is may be due to the regular and low prescription of these antibiotics. Epidemiological investigations of Nigeria, Senegal, Iran, India, China, Taiwan, Saudi-Arabia, Thailand, Egypt, Brazil, Colombia and Argentina showed that the H. pylori isolates of human clinical specimens had the highest levels of resistance against metronidazole, amoxicillin, quinolones and tetracycline [39] which was similar to our results.
The results of antimicrobial resistance pattern had indirectly confirmed that the H. pylori strains of hamburger, soup and olvie salad (meat based ready to eat foods) were transferred from the meat samples of infected animals. It is because of the H. pylori strains of these food stuffs were relatively resistant to the antibiotics used especially in the veterinary medicine. Prescription of metronidazole, clarithromycin, levofloxacin, rifampin, cefsulodin, furazolidone and spiramycin antimicrobial agents is not routine in the cases of diseases in animals. Therefore, the prevalence of resistance against these antibiotics in the H. pylori strains of hamburger, soup and olvie salad samples which are relatively made from meat is low and this finding can indirectly confirm the primary infections of meat used from production of hamburger, soup and olvie salad.

Conclusions
In conclusion, ready to eat foods in Iran harbor H. pylori strains with high prevalence of vacA, cagA, iceA and oipA genotypes. High prevalence of H. pylori in our samples suggest that contaminated ready to eat foods maybe the sources of the bacteria and that it entered the human population in period of time. Diversity of H. pylori