Genotyping of the Helicobacter pylori isolates of raw milk and traditional dairy products

Notwithstanding the substantial clinical impact of Helicobacter pylori, its convinced routes of transmission and sources have not been reported. Based on the quarrelsome hypothesis, foods and especially dairy products play an authoritative role in the transmission of H. pylori to humans. The current investigation was done to study the prevalence rate and distribution of vacA genotypes in the H. pylori strains isolated from the raw milk and traditional dairy products. Three-hundred milk and dairy samples were collected and directly transported to laboratory. Samples were cultured and H. pylori isolates were approved using the 16s rRNAbased PCR amplification. Positive strains were tested for distribution of vacA genotypes using the multiplex-PCR. Sixty out of 300 samples (20%) harbored H. pylori. Prevalence of H. pylori in milk and traditional dairy products were 38.75% and 13.18%, respectively. Ovine milk (45%) and traditional cheese (40%) had the highest prevalence of H. pylori. VacAs1a (91.66%), vacAm1a (61.61%) vacAs2 (36.66%) and vacAm2 (31.66%) were the most commonly detected genotypes. Ovine milk and traditional cheese had the most diverse genotypes. S1am1a (41.66%), s2m1a (25%), s1am2 (16.66%) and s2m2 (13.33%) were the most commonly detected combined genotypes. Raw milk and traditional dairy products are latent sources of H. pylori. Similarity in the genotyping pattern of H. pylori strains of various samples represents their similar sources of infection. Further studies are required to found the exact sources of H. pylori strains in raw milk and traditional dairy products.


Introduction
The contamination of food with microbes can happen at any step of the food chain.Milk is virtually sterile when it is synthesized in a healthy animal's udder.Ruminants are natural reservoirs of bacteria.Many of these bacteria are not harmful to humans, but some may be harmful to humans even though the animals are not affected and appear healthy.The extremely nourishing nature of dairy products makes them particularly suitable media for bacterial proliferation.Raw milk and dairy products can harbor a variety of pathogenic organisms. 1hough Helicobacter pylori (H.3][4] Suitable circumstances for growth and survival of H. pylori in milk and dairy products provide adequate settings for transmission of H. pylori from these foodstuffs to human. 5he severity of clinical complications caused by H. pylori is depends on several factors.Presence of pathogenic virulence genes and genotypes the most important factor responsible for H. pylori infections.Vacuolating cytotoxin (vacA) is one of the most important virulence factors in the occurrence of human clinical diseases caused by this bacterium.The vacA belongs to the group of genes with variable genotypes or structures.The vacA gene is present in virtually all strains of H. pylori but it is polymorphic, comprising variable signal regions (type s1 or s2) and mid-regions (type m1 or m2).The s1 type is additional subtyped into s1a, s1b and s1c subtypes, and the m1 into m1a and m1b subtypes. 6,7he mosaic combination of s and m-region allelic types determines the particular cytotoxin and, consequently, the pathogenicity of the bacterium.Genotyping using vacA virulence marker is considered as one of the best approaches for study of correlations between H. pylori isolates from different samples. 6,7

Sample collection
From May 2015 to July 2015, overall 300 dairy products including raw bovine milk (n=40), raw ovine milk (n=40), traditional cheese (n=50), traditional cream (n=50), traditional butter (n=60) and tradi-tional ice-cream (n=60) were purchased from the supermarkets of Tehran province, Iran.Samples (100 mL, in sterile glass containers) were transported to the laboratory at 4°C.All samples were kept under refrigeration in plastic bags; information about dates of production and of assigned shelf lives was not presented.

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'-CTGGAGAGAC-TAAGCCCTCC-3' and HP-R: 5'-ATTACT-GACGCTGATTGTGC-3') (110 bp). 8PCR 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 picomole of each primer.PCR was performed using a thermal cycler (Eppendorf Co., Germany) under the following conditions: an initial denaturation for 2 minutes 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.

Genotyping of vacA genes of Helicobacter pylori
Presence of the vacA (s1a, s1b, s1c, m1a, m1b and m2) alleles were determined using PCR technique.Primers, PCR conditions and volume of each reaction are shown in Table 1. 9 PCR amplifications were performed using a DNA thermal cycler (Eppendorf Co., Germany).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).
Table 1.Oligonucleotide primers and PCR conditions used for genotyping of Helicobacter pylori strains isolated from dairy products. 9

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 dairy products were statistically analyzed.

Results
Table 2 represents the total prevalence of H. pylori isolated from different types of dairy products.All of the H. pylori isolates harbored the 16SrRNA gene in the PCR amplification (Figure 1).We found that 60 out of 300 samples (20%) were positive for the H. pylori.Prevalence of H. pylori among milk and traditional dairy products were 38.75% and 13.18%, respectively.Ovine milk (45%) and traditional cheese (40%) had the highest prevalence of H. pylori strains.Statistically significant differences were seen between the type of samples and prevalence of H. pylori strains (P<0.05).
Table 3 represents the distribution of vacA genotypes among H. pylori isolates of dairy products.VacAs1a (91.66%), m1a (61.61%) s2 (36.66%) and m2 (31.66%) were the most commonly detected genotypes amongst the H. pylori isolates.Ovine milk and traditional cheese had the most diverse genotypes.VacAs1c (5%) had the lowest prevalence amongst the H. pylori isolates of all dairy samples.Statistically significant differences were seen between the type of samples and prevalence of different genotypes (P<0.05).
Table 4 represents the distribution of combined genotypes of the H. pylori strains isolated from all studied samples.We found that s1am1a (41.66%), s2m1a (25%), s1am2 (16.66%) and s2m2 (13.33%) were the most commonly detected genotypes among the H. pylori isolates of all dairy products.There were no positive results for s1cm1b and s1cm2 combined genotypes.

Discussion
Results of the present study revealed that 20% of dairy products were contaminated with H. pylori strains which showed an important public health threat regarding the consumption of raw milk and traditional dairy products.Results also indicated the high distribution of putative genotypes in the H. pylori isolates of milk and dairy products.
Our results showed that ovine milk samples had the highest prevalence of H. pylori.This substance has been accepted by other researchers. 10,11,13,16,19It is maybe due to the high ability of the sheep stomach to keep H. pylori and then its transmission into the environment.The main reason for the high prevalence of H. pylori in dairy samples is the fact that milk has an appropriate conditions and especially pH and activated water (AW) which support the growth and survival of H. pylori strains.Traditional cheese had also high prevalence of H. pylori and it is maybe due to the fact that the milk which used for preparation of cheese should not heat higher than 40°C.Therefore, H. pylori and also other types of bacteria can survive in this type of traditional dairy.
VacAs1a, m1a, s2 and m2 genotypes had a considerable prevalence in H. pylori strains.Similar findings have been reported previously in milk, 10,16,19 meat, 9,11,16 vegetable 9 and ready to eat foods. 20emmatinezhad et al. (2016) 21 reported that the prevalence of H. pylori in various types of ready to eat food samples were 13.45%.They showed that olvie salad (36%), restaurant salad (30%), fruit salad (28%) and soup (22%) had the highest prevalence rate.Their findings reported that the most commonly detected combined genotypes were s1am2 (70.27%), s1am1a (39.18%) and m1am2 (31.08%) which was similar to our findings.Yahaghi et al. (2014) 9 revealed that cagA (57.62%), vacA s1a (37.28%) and vacA m1a (30.50 %) had the highest prevalence among the H. pylori strains of vegetables.High prevalence of vacA genotypes among the clinical isolates and cases of gastrointestinal disorders have been reported from Iran, 22 United States, 23 Australia, 24 United Kingdom 25 and China. 26djacent connotation of vacA genotypes with production of interleukin 8 (IL-8) and cytotoxins, adhesion to gastric epithelial cells, occurrence of inflammation, vacuolization, necrosis and apoptosis of epithelial cells has been reported in previously published data. 27,28High prevalence of these genotypes in milk and dairy samples of our investigation showed their high pathogenic nature.

Article
reference strain of H. pylori (ATCC 43504) was employed.