# vacA Genotype Status of Helicobacter pylori Isolated from Foods with Animal Origin

Authors: Elnaz Saeidi; Amirhossein Sheikhshahrokh
Journal: BioMed Research International
 (2016)

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## Abstract

According to controversial theories and results of studies, foods with animal origins play an important role in the transmission ofH. pylori to human. The aim of this study was to determine the distribution ofvacA genotypes ofH. pylori, isolated from milk and meat samples of cow, sheep, goat, camel, and buffalo. Eight hundred and twenty raw milk and meat samples were collected from various parts of Iran. Samples were cultured and those found positive forH. pylori were analyzed for the presence of various genotypes ofvacA gene. Out of 420 milk and 400 meat samples, 92 (21.90%) and 105 (26.25%) were positive forH. pylori, respectively. The most commonly detected genotypes in thevacA gene weres1a (86.80%),m1a (79.18%),s1b (69.54%), andm1b (63.45%) and detected combined genotypes were mostlym1as1a (68.52%),m1as1b (60.40%),m1bs1b (55.83%), andm1bs1a (53.29%). High presence of bacteria in the milk and meat samples of sheep represents that sheep may be the natural host ofH. pylori. High presence ofH. pylori strains in milk and meat samples similar tovacA genotypes in human being suggests that milk and meat samples could be the sources of bacteria for human.


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## Body

## 1. Introduction

AlthoughHelicobacter pylori (H. pylori) has been accepted as a major cause of gastrointestinal disorders and especially gastric adenocarcinoma, type B gastritis, mucosa associated lymphoid tissue lymphoma, and peptic ulcer disease, its route of transmission, sources, and also the role of foods are still unknown.H. pylori is a Gram negative, coccoid flagellated bacterium with 2 to 4 μm in length and 0.5 to 1 μm in width, which is the first formally recognized bacterial carcinogen and one of the most successful human pathogens [1–5]. High prevalence ofH. pylori in the stomach of domestic animals, milk, meat, and gastric biopsies suggests that food with animal origin, and also domestic animals, may be its reservoirs [1–5]. Higher prevalence ofH. pylori in meat eaters than vegetarians which was achieved in the previous investigation supports the significant role of foods with animal origins in the transmission of bacteria to humans [6]. Appropriate condition of meat and milk including acidic PH, nutritional values, salt concentration, and also high amount of activated water (AW) facilitate the growth and survival ofH. pylori and provide adequate setting for transmission ofH. pylori to human [7]. High prevalence of antibodies againstH. pylori in the serum samples taken from veterinarians, butchers, and staffs of the slaughterhouses and milking rooms can support the zoonoses aspects of this bacterium [1, 8].To evaluate the pathogenicity ofH. pylori, apprising virulence factors is requisite. The most commonly identified virulence factor amongH. pylori strains is vacuolating cytotoxin (vacA) [9, 10].VacA belongs to the group of genes with mutable genotypes associated with damage to gastric epithelial cells. This gene exists in practically all strains ofH.pylori. This gene is polymorphic and comprises variable signal regions (types1 or types2) and midregions (typem1 or typem2) [9, 10]. The s-region is classified intos1 ands2 and the m-region is categorized asm1 andm2. Thes1 type is further subtyped intos1a,s1b, ands1c and the subcategories ofm1 arem1a andm1b, respectively. Higher cytotoxicity and acuity have been done by this mosaic pattern [11, 12]. Genotyping usingvacA alleles is considered as one of the best methods to study the associations ofH.pylori strains in various samples.Industrialized information designated the fact that closely 50% of the world population and also 60–90% of Iranian people are infected with virulent strains ofH. pylori [4, 13]. According to the high prevalence ofH. pylori in Iran and other parts of the world, and also with respect to the indistinct situation ofH. pylori in foods with animal origins, the present investigation was carried out in order to study thevacA genotype status ofH. pylori isolated from Iranian raw milk and meat samples.


## 2. Materials and Methods

### 2.1. Sample Collection

In all, 420 raw milk samples were collected: cow (n
=
120), sheep (n
=
120), goat (n
=
80), buffalo (n
=
50), and camel (n
=
50) raw milk samples were collected from farm bulk tanks and milk collection centers from several geographic regions of Iran, from March 2013 to March 2014. Cow and buffalo milk samples were collected throughout this time period. Because the lactating periods of ewes and goats in Iran are seasonal (from March through May and September to November of the subsequent year), goat and sheep milk samples were only available through these months within the fore-mentioned time frame. At each site, sampling of milk was performed according to the International Dairy Federation guidelines (IDF 1995). Samples (100 mL, in sterile glass containers) were transported to the laboratory at ca. 4°C within a maximum of 6–12 h after sampling. For raw meat samples, 100 cow, 100 sheep, 100 goat, 50 buffalo, and 50 camel meat samples were purchased from butchers of various parts of Iran. All samples were kept under refrigeration in plastic bags; information about dates of production and assigned shelf lives was not presented. Meat samples were collected over a period of eight months from August 2013 to February 2014, and they were analyzed on the day of acquisition. Samples were transported under refrigeration (4–6°C) in thermal boxes containing ice packs and were tested immediately after collection.


### 2.2. Isolation ofHelicobacter pylori

Twenty-five mL of each homogenized sample was added to 225 mL of Wilkins Chalgren anaerobe broth (Oxoid, UK) supplemented with 5% of horse serum (Sigma, St. Louis, MO, USA) and colistin methanesulfonate (30 mg/L), cycloheximide (100 mg/L), nalidixic acid (30 mg/L), trimethoprim (30 mg/L), and vancomycin (10 mg/L) (Sigma, St. Louis, MO, USA) and colistin methanesulfonate (30 mg/L), cycloheximide (100 mg/L), nalidixic acid (30 mg/L), trimethoprim (30 mg/L), and vancomycin (10 mg/L) (Sigma, St. Louis, MO, USA) and incubated for 7 days at 37°C with shaking under microaerophilic condition. Then, 0.1 mL of the enrichment selective broth was plated onto Wilkins Chalgren anaerobe agar (Oxoid, UK) supplemented with 5% of defibrinated horse blood and 30 mg/L colistin methanesulfonate, 100 mg/L cycloheximide, 30 mg/L nalidixic acid, 30 mg/L trimethoprim, and 10 mg/L vancomycin (Sigma, St. Louis, MO, USA) and incubated for 7 days at 37°C under microaerophilic condition. For comparison, a reference strain ofH. pylori (ATCC 43504) was employed.


### 2.3. DNA Extraction andHelicobacter pylori 16S rRNA Gene Amplification

Based on the PCR technique, suspected colonies were identified asH. pylori. Genomic DNA was extracted from the colonies with typical characters ofH. pylori using a DNA extraction kit for cells and tissues (Roche Applied Science, Germany, 11814770001) according to the manufacturer’s instructions and its density which 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 10x buffer + MgCl2, 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 condition: 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.


### 2.4. Genotyping ofvacA Gene ofHelicobacter pylori

Presence of the genotypes ofvacA alleles (s1a,s1b,s1c,m1a,m1b, andm2) was determined by PCR. The primer sequences are shown in Table 1 [15].Table 1
Oligonucleotide primers used for genotyping ofHelicobacter pylori isolated from foods with animal origin in Iran [15].
vacA alleles
Primer sequence (5′-3′)
Size of product (bp)
s1a
F: CTCTCGCTTTAGTAGGAGCR: CTGCTTGAATGCGCCAAAC
213
s1b
F: AGCGCCATACCGCAAGAGR: CTGCTTGAATGCGCCAAAC
187
s1c
F: CTCTCGCTTTAGTGGGGYTR: CTGCTTGAATGCGCCAAAC
213
s2
F: GCTAACACGCCAAATGATCCR: CTGCTTGAATGCGCCAAAC
199
m1a
F: GGTCAAAATGCGGTCATGGR: CCATTGGTACCTGTAGAAAC
290
m1b
F: GGCCCCAATGCAGTCATGGAR: GCTGTTAGTGCCTAAAGAAGCAT
291
m2
F: GGAGCCCCAGGAAACATTGR: CATAACTAGCGCCTTGCA
352The PCR was performed in a total volume of 50μL containing 1 μM of each primer, 1 μL of genomic DNA (approximately 200 ng), 1 mM of dNTPs mix (invitrogen), 2 mM of M
g
c
l
2, 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: 32 cycles of 45 s at 95°C, 50 s at 64°C, and 70 s at 72°C. All runs included one negative DNA control consisting of PCR grade water and two or more positive controls (H. pylori 26695,H. pylori J99,H. pylori SS1,H. pylori Tx30,H. pylori 88-23, andH. pylori 84-183).


### 2.5. Gel Electrophoresis

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


### 2.6. Statistical Analysis

Using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA), Chi-square test and Fisher’s exact two-tailed test analysis were performed, and differences were considered significant at values ofP
<
0.05. Distributions of genotypes ofH. pylori isolated from food stuff were statistically analyzed.


## 2.1. Sample Collection

In all, 420 raw milk samples were collected: cow (n
=
120), sheep (n
=
120), goat (n
=
80), buffalo (n
=
50), and camel (n
=
50) raw milk samples were collected from farm bulk tanks and milk collection centers from several geographic regions of Iran, from March 2013 to March 2014. Cow and buffalo milk samples were collected throughout this time period. Because the lactating periods of ewes and goats in Iran are seasonal (from March through May and September to November of the subsequent year), goat and sheep milk samples were only available through these months within the fore-mentioned time frame. At each site, sampling of milk was performed according to the International Dairy Federation guidelines (IDF 1995). Samples (100 mL, in sterile glass containers) were transported to the laboratory at ca. 4°C within a maximum of 6–12 h after sampling. For raw meat samples, 100 cow, 100 sheep, 100 goat, 50 buffalo, and 50 camel meat samples were purchased from butchers of various parts of Iran. All samples were kept under refrigeration in plastic bags; information about dates of production and assigned shelf lives was not presented. Meat samples were collected over a period of eight months from August 2013 to February 2014, and they were analyzed on the day of acquisition. Samples were transported under refrigeration (4–6°C) in thermal boxes containing ice packs and were tested immediately after collection.


## 2.2. Isolation ofHelicobacter pylori

Twenty-five mL of each homogenized sample was added to 225 mL of Wilkins Chalgren anaerobe broth (Oxoid, UK) supplemented with 5% of horse serum (Sigma, St. Louis, MO, USA) and colistin methanesulfonate (30 mg/L), cycloheximide (100 mg/L), nalidixic acid (30 mg/L), trimethoprim (30 mg/L), and vancomycin (10 mg/L) (Sigma, St. Louis, MO, USA) and colistin methanesulfonate (30 mg/L), cycloheximide (100 mg/L), nalidixic acid (30 mg/L), trimethoprim (30 mg/L), and vancomycin (10 mg/L) (Sigma, St. Louis, MO, USA) and incubated for 7 days at 37°C with shaking under microaerophilic condition. Then, 0.1 mL of the enrichment selective broth was plated onto Wilkins Chalgren anaerobe agar (Oxoid, UK) supplemented with 5% of defibrinated horse blood and 30 mg/L colistin methanesulfonate, 100 mg/L cycloheximide, 30 mg/L nalidixic acid, 30 mg/L trimethoprim, and 10 mg/L vancomycin (Sigma, St. Louis, MO, USA) and incubated for 7 days at 37°C under microaerophilic condition. For comparison, a reference strain ofH. pylori (ATCC 43504) was employed.


## 2.3. DNA Extraction andHelicobacter pylori 16S rRNA Gene Amplification

Based on the PCR technique, suspected colonies were identified asH. pylori. Genomic DNA was extracted from the colonies with typical characters ofH. pylori using a DNA extraction kit for cells and tissues (Roche Applied Science, Germany, 11814770001) according to the manufacturer’s instructions and its density which 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 10x buffer + MgCl2, 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 condition: 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.


## 2.4. Genotyping ofvacA Gene ofHelicobacter pylori

Presence of the genotypes ofvacA alleles (s1a,s1b,s1c,m1a,m1b, andm2) was determined by PCR. The primer sequences are shown in Table 1 [15].Table 1
Oligonucleotide primers used for genotyping ofHelicobacter pylori isolated from foods with animal origin in Iran [15].
vacA alleles
Primer sequence (5′-3′)
Size of product (bp)
s1a
F: CTCTCGCTTTAGTAGGAGCR: CTGCTTGAATGCGCCAAAC
213
s1b
F: AGCGCCATACCGCAAGAGR: CTGCTTGAATGCGCCAAAC
187
s1c
F: CTCTCGCTTTAGTGGGGYTR: CTGCTTGAATGCGCCAAAC
213
s2
F: GCTAACACGCCAAATGATCCR: CTGCTTGAATGCGCCAAAC
199
m1a
F: GGTCAAAATGCGGTCATGGR: CCATTGGTACCTGTAGAAAC
290
m1b
F: GGCCCCAATGCAGTCATGGAR: GCTGTTAGTGCCTAAAGAAGCAT
291
m2
F: GGAGCCCCAGGAAACATTGR: CATAACTAGCGCCTTGCA
352The PCR was performed in a total volume of 50μL containing 1 μM of each primer, 1 μL of genomic DNA (approximately 200 ng), 1 mM of dNTPs mix (invitrogen), 2 mM of M
g
c
l
2, 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: 32 cycles of 45 s at 95°C, 50 s at 64°C, and 70 s at 72°C. All runs included one negative DNA control consisting of PCR grade water and two or more positive controls (H. pylori 26695,H. pylori J99,H. pylori SS1,H. pylori Tx30,H. pylori 88-23, andH. pylori 84-183).


## 2.5. Gel Electrophoresis

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


## 2.6. Statistical Analysis

Using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA), Chi-square test and Fisher’s exact two-tailed test analysis were performed, and differences were considered significant at values ofP
<
0.05. Distributions of genotypes ofH. pylori isolated from food stuff were statistically analyzed.


## 3. Results and Discussion

All of the milk and meat samples were examined using the culture and PCR techniques. Table2 shows the distribution ofH. pylori in the milk and meat samples. Of 820 meat and milk samples, 197 (24.02%) were positive forH. pylori. Of 420 milk and 400 meat samples, 92 (21.90%) and 105 (26.25%) were positive forH. pylori, respectively (Table 2).Table 2
Distribution ofHelicobacter pylori in various types of raw milk and meat samples.
Types of samples
Number of samples collected
Positive results forH. pylori (%)
Cow milk
120
25 (20.83)
Sheep milk
120
35 (29.16)
Goat milk
80
15 (18.75)
Buffalo milk
50
12 (24)
Camel milk
50
5 (10)
Total raw milk
420
92 (21.90)
Cow meat
100
25 (25)
Sheep meat
100
37 (37)
Goat meat
100
22 (22)
Buffalo meat
50
14 (28)
Camel meat
50
7 (14)
Total raw meat
400
105 (26.25)
Total
820
197 (24.02)The most commonly contaminated milk and meat samples were raw sheep milk (29.16%) and raw sheep meat (37%). There were no statistically significant differences among the incidence of bacteria in milk and meat samples. There were statistically significant differences in the incidence ofH. pylori between sheep and camel milk (P
=
0.033) and between sheep and camel meat (P
=
0.048). Distribution ofvacA genotypes of theH. pylori strains of meat and milk samples is shown in Table 3.Table 3
Distribution ofvacA genotypes in Helicobacter pylori strains of meat and milk samples.
Types of samples (number of positive results)
Distribution ofvacA genotypes (%)
s1a
s1b
s1c
s2
m1a
m1b
m2
Cow milk (25)
20
18
14
9
19
17
8
Sheep milk (35)
32
23
16
12
27
20
10
Goat milk (15)
12
10
8
5
10
9
4
Buffalo milk (12)
10
8
5
7
8
8
2
Camel milk (5)
5
4
—
2
3
3
—
Total raw milk(92)
79 (85.86)
63 (68.47)
43 (46.73)
35 (38.04)
67 (72.82)
57 (61.95)
24 (26.08)
Cow meat (25)
22
19
14
10
20
19
9
Sheep meat (37)
34
26
20
13
29
21
13
Goat meat (22)
19
15
11
8
17
16
7
Buffalo meat (14)
11
9
6
9
10
9
5
Camel meat (7)
6
5
1
3
3
3
1
Total raw meat(105)
92 (87.61)
74 (70.47)
52 (49.52)
43 (40.95)
89 (84.76)
68 (64.76)
35 (33.33)
Total(197)
171 (86.80)
137 (69.54)
95 (48.22)
78 (39.59)
156 (79.18)
125 (63.45)
59 (29.94)Results of the gel electrophoresis of PCR products for amplification of various genotypes ofvacA gene are shown in Figures 1–3.Figure 1
Results of the gel electrophoresis for identification ofm2,s1b, ands1c genotypes of theH. pylori strains of milk and meat samples. Line 5: negative control, line 7: positive controls, 1: negative sample, M: 100 bp DNA ladder (Fermentas, Germany), and numbers 2–4: positive samples form2 (352 bp),s1b (187 bp), ands1c (213 bp) alleles.Figure 2
Results of the gel electrophoresis for identification ofm1a ands1a genotypes of theH. pylori strains of milk and meat samples. Lanes 3 and 4: positive controls, 5: negative control, M: 100 bp DNA ladder (Fermentas, Germany), and numbers 1-2: positive samples form1a (290 bp) ands1a (213 bp) alleles, respectively.Figure 3
Results of the gel electrophoresis for identification ofm1b ands2 genotypes of theH. pylori strains of milk and meat samples. Lanes 3 and 4: positive controls, 5: negative control, M: 100 bp DNA ladder (Fermentas, Germany), and numbers 1-2: positive samples fors2 (199 bp) andm1b (291 bp) alleles, respectively.Significant difference was found between the type of samples and prevalence of genotypes (P
<
0.05). Fourteen different genotypic combinations are shown in Table 4. The most commonly detected combined genotypes werem1as1a (68.52%),m1as1b (60.40%),m1bs1b (55.83%), andm1bs1a (53.29%).Table 4
Distribution of combined genotypes ofHelicobacter pylori isolated from Iranian raw milk and meat samples.
Genotypes
Prevalence (%)∗
M1as1a
135 (68.52)
M1as1b
119 (60.40)
M1bs1a
105 (53.29)
M1bs1b
110 (55.83)
M1as1c
70 (35.53)
M1bs1c
58 (29.44)
M2s1a
40 (20.30)
M2s1b
31 (15.73)
M2s1c
26 (13.19)
M2s2
19 (9.64)
M1as2
56 (28.42)
M1bs2
44 (22.33)
∗Percent of positive genes from total of 197 positive samples.Results of current study showed that raw milk and meat samples were reservoir forH. pylori. Total prevalences ofH. pylori in raw cow, sheep, goat, buffalo, and camel milk samples of our survey were 20.83%, 29.16%, 18.75%, 24%, and 10%, respectively. Rahimi and Kheirabadi [3] reported that the incidence ofH. pylori in raw cow, sheep, goat, buffalo, and camel milk samples of Iranian herds was 1.41%, 12.20%, 8.70%, 23.4%, and 3.6%, respectively, which was lower than our results. In a study carried out in Italy,H. pylori was detected in 50%, 33%, and 25.6% of raw cow, sheep, and goat milk, respectively, which was higher than our results [16]. In a study conducted in Japan,H. pylori was detected in 72.2% of raw cow milk samples [17]. Total distribution ofH. pylori in the milk samples of Greek [18] and American [4] herds was 20% and 60%, respectively. Recent clinical investigation among Iranian cows showed that 16% of milk and 40% of feces samples of seropositive herds were infected withH. pylori [2].Total prevalence ofH. pylori in cow, sheep, goat, buffalo, and camel meat samples of our survey was 25%, 37%, 22%, 28%, and 14%, respectively, which was entirely contrary to the results of Stevenson et al. [19]. They suggested that transmission ofH. pylori from beef and beef products is not a primary factor in the high prevalence of this bacterium in humans. Moreover, Mhaskar et al. [20] reported that the prevalence of peptic ulcer andH. pylori infection were entirely higher in those patients who have used meat and meat products (Odds Ratio (OR): 2.35, 95% and Confidence Interval (CI): 1.30–4.23) and restaurant foods (OR: 3.77, 95% CI: 1.39–10.23) in their main meals.The possibility thatH. pylori may be a zoonosis first rose the publication of two epidemiological studies that exhibited that the prevalence ofH. pylori infection in abattoir and meat workers was significantly increased in comparison with the subjects that were not involved in handling animals or meat [21, 22]. This hypothesis is further reinforced by the demonstration ofH. pylori in the gastric mucosa of calves, pigs, and horses and its isolation from sheep’s gastric tissue and milk [4], suggesting that these animal species may act as reservoirs and spreaders ofH. pylori. Findings of Momtaz et al. [1] have conclusively proved the zoonotic aspects ofH. pylori. They showed that thevacA s1a/m1a was prominentH. pylori genotype in all cow, sheep, and human beings clinical samples. They showed 3.4–8.4% variability and 92.9–98.5% homology between sheep and human samples [1].High prevalence ofH. pylori in the milk (29.16%) and meat (37%) samples of sheep of our study suggested that sheep may be the natural host ofH. pylori. Dore et al. [4] reported thatH. pylori DNA was demonstrated in 60% (38/63) of milk samples and in 30% (6/20) of sheep tissue samples. They showed that thevacA gene was amplified in five of 38 milk samples, and in two of six sheep tissue samples, respectively. Sequence analysis of 16S rRNA PCR products fromH. pylori strains [4] investigation demonstrated 99% identity withH. pylori.Genotyping usingvacA virulence marker gene is considered as one of the best approaches for studying of the correlations betweenH.pylori isolates from different samples [1].H. pylori strains of milk and meat samples of our study showed similar status in the distribution ofvacA genotypes. Totally, the most commonly detected genotypes in milk and meat samples weres1a (86.80%),m1a (79.18%),s1b (69.54%), andm1b (63.45%). Various genotypes ofvacA strains were the most commonly detected genotypes in the studies of Linpisarn et al. (Thailand) [23], López-Vidal et al. (Mexico) [24], and Rudi et al. (Germany) [25]. The high presence ofm1as1a andm1as1b genotypes has been reported previously from Iran [13] and Germany [25] but far different results have been reported from Thailand [23] and Mexico [24].According to the high prevalence of pathogenic strains ofH. pylori in milk and meat samples especially in those collected from sheep and also based on the considerable consumption of milk and meat in their raw forms in some areas of the world [26–28], consumption of these food products in their raw forms should be stopped. On the other hand, thorough cooking of meat and pasteurization of milk can prevent the presence and also transmission of pathogenic bacteria likeH. pylori.


## 4. Conclusions

H. pylori which is harbored from milk and meat samples are similar in genotype of thevacAallele with isolates recovered from human. Also, since there was a high similarity in the genotyping pattern ofH. pylori DNA among milk and meat samples and human specimens of other investigations, it is suggested that raw milk and meat samples are the sources of the bacteria and that they entered the human population in period of time. On the other hand, diversity ofH. pylori genotypes between milk and meat samples with the clinical isolation of other studies suggested that consumption of contaminated milk and meat withH. pylori strains may be a threat to human health.



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*Source: 8701067-2016-03-07.xml*