Phenotypic analysis of antibiotic resistance and genotypic study of the vacA, cagA, iceA, oipA and babA genotypes of the Helicobacter pylori strains isolated from raw milk

Background Foods with animal origins and particularly milk play a considerable role in transmission of Helicobacter pylori. The current study was performed to assess phenotypic characters of antibiotic resistance and genotyping pattern of vacA, cagA, iceA, oipA and babA2 alleles amongst the H. pylori strains isolated from raw milk. Methods Six-hundred and thirty raw milk samples were collected and cultured on Wilkins Chalgren anaerobe media. Antibiotic resistance and genotyping patterns were studied using disk diffusion and PCR, respectively. Results Sixty-seven out of 630 (10.63%) raw milk samples were positive for H. pylori. Ovine raw milk (17.27%) samples had the highest prevalence of H. pylori, while camel (5.00%) had the lowest. H. pylori strains harbored the highest prevalence of resistance against ampicillin (82.08%), tetracycline (76.11%), amoxicillin (74.62%), metronidazole (65.67%) and erythromycin (53.73%). Prevalence of resistance against more than 10 types of antibiotics was 17.91%. VacA s1a (83.58%), m1a (80.59%), s2 (77.61%) and m2 (68.65%), cagA (73.13%) and babA2 (44.77%) were the most commonly detected genotypes. We found that S1am1a (56.71%), s2m1a (56.71%), s1 am2 (43.28%) and s2 m2 (43.28%) were the most commonly detected genotyping pattern. Frequency of cagA-, oipA- and babA2- genotypes were 26.86%, 62.68% and 55.22%, respectively. We found that S1a/cagA+/iceA1/oipA−/babA2- (28.35%), m1a/cagA+/iceA1/oipA−/babA2- (28.35%) and s2/cagA+/iceA1/oipA−/babA2- (26.86%) were the most commonly detected combined genotyping pattern. Conclusions Simultaneous presence of vacA, cagA, iceA, oipA and babA2 genotypes in antibiotic resistant H. pylori strains indicates important public health issue regarding the consumption of raw milk. However, additional researches are required to find molecular genetic homology and other epidemiological aspects of H. pylori in milk.


Background
Milk of animals provide a package of key nutrients that are difficult to obtain in diets with limited or no dairy products [1]. Dissimilarly, raw milk is not necessarily safe, as evidenced by higher rates of foodborne illnesses associated with its consumption [2][3][4][5][6]. Likewise, there were so many investigations about the considerable prevalence of some specific foodborne pathogens in raw milk [2][3][4][5][6].
Helicobacter pylori (H. pylori) is a microaerophilic and Gram-negative spiral coccoid flagellated bacterium with 2 to 4 μm in length and 0.5 to 1 μm in width. It is known as one of the main causative agents of duodenal ulcer, peptic ulcer disease, gastric adenocarcinoma, type B gastritis and gastric B-cell lymphoma [7][8][9]. Human stomach is considered as a main reservoir of H. pylori strains [7][8][9]. In keeping with this, foods with animal origins may play an imperative role in transmission of H. pylori infections to human [7][8][9]. Suitable conditions including pH, activated water (AW), moisture and temperature cause H. pylori to easily survive in milk [10]. Raw milk [10], pasteurized milk [7][8][9] and even sterilized food samples [10] have been introduced as possible emerging sources of H. pylori infections. Vacuolating Cytotoxin A (vacA) and Cytotoxin Associated Gene A (cagA) are two important virulence genes with high importance in the pathogenicity of H. pylori infections [7][8][9][10]. The vacA gene is polymorphic, comprising variable signal regions (type s1 or s2) and mid-regions (type m1 or m2). The s1 type is additionally divided into s1a, s1b and s1c and the m1 into m1a and m1b subtypes. The cagA gene has been detected in the severe cases of gastrointestinal disorders and peptic ulcers [7][8][9][10]. Induced by contact with the epithelium antigen (iceA), outer inflammatory protein (oip) and blood group antigen-binding adhesin gene (babA) are other important pathogenic genotypes of the H. pylori strains [7][8][9][10][11]. Genotyping using these virulence markers is considered as one of the best approaches to study the correlations between H. pylori isolates from different samples [11].
Antibiotic therapy is one of the best aspects of treatments for H. pylori infections. However, therapeutic options have become somewhat restricted because of the presence of severe resistance in some strains of this bacterium [12]. Documented data disclosed that H. pylori strains harbored the high prevalence of resistance against different types of antibiotics [12].
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 infections such as Iran [7-10, 13, 14]. Considering the indistinct epidemiological aspects of H. pylori in milk and due to the high prevalence of H. pylori all-around the world [7][8][9][10][11][12][13][14], the present investigation was performed in order to study the prevalence rate, genotyping patterns and phenotypic evaluation of antibiotic resistance of the H. pylori strains isolated from raw milk samples of bovine, ovine, caprine, buffalo and camel.

Samples
From January to March 2018, total 630 raw milk samples of bovine (n = 120), ovine (n = 110), caprine (n = 130), buffalo (n = 130) and camel (n = 140) were arbitrarily collected from the supermarkets of diverse areas of Isfahan province, Iran. All milk samples were collected from traditional dairy farms. Milk samples were kept at refrigerator. Throughout milk collection, the first few squirts were overlooked. The animals which their milk samples collected for this research were clinically healthy, and the milk samples displayed natural physical (color, odor, pH, and density) constancy. Samples (50 ml, in sterile glass bottles) were transported in ice-cooled flasks (at 4°C) to the laboratory within two hours after collection.

Isolation of helicobacter pylori
Isolation of H. pylori bacteria was performed using the culture technique [7-10, 13, 14]. Twenty-five milliliters of milk sample were used for this purpose. Wilkins Chalgren anaerobe broth (Oxoid Ltd., Basingstoke, UK) was used for this purpose. Microaerophilic conditions (5% oxygen, 85% nitrogen and 10% CO 2 ) was prepared using the MART system (MART system, Lichtenvoorde, The Netherland).

DNA extraction and 16S rRNA-based PCR confirmation
Distinctive colonies of H. pylori were additionally approved using the 16S rRNA-based PCR method. Typical colonies were sub-cultured on Wilkins Chalgren anaerobe broth supplemented with same materials mentioned above [15]. Genomic DNA was then extracted from colonies using a DNA extraction kit (Thermo Fisher Scientific, St. Leon-Rot, Germany). Procedure was performed rendering to the manufacturer's guidelines. Purity (A260/A280) and concentration of extracted DNA were then checked (NanoDrop, Thermo Scientific, Waltham, MA, USA). The truth of the DNA was assessed on a 2% agarose gel stained with ethidium bromide (0.5 μg/mL) (Thermo Fisher Scientific, St. Leon-Rot, Germany). Polymerase Chain Reaction (PCR) was performed using a PCR thermal cycler (Eppendorf Co., Hamburg, Germany) according to reported procedure [15].

Genotyping analysis
Frequency of vacA, cagA, iceA, oipA and babA alleles were assessed using PCR [19][20][21][22]. Table 1 characterizes the set of primers and PCR circumstances applied for genotyping of vacA, cagA, iceA, oipA and babA alleles. Initially, all samples were subjected to pre-tests to found suitable time, temperature and volume of reaction. A programmable DNA thermo-cycler (Eppendorf Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany) was used in all PCR reactions. PCR grade water and H. pylori standard strains (SS1, 26,695, Tx30, J99, 88-23 and 84-183) were used as negative and positive controls, respectively. Ten microliters of PCR product were exposed to electrophoresis in a 2% agarose gel in 1X TBE buffer at 80 V for 30 min, stained with SYBR Green. The UVI doc gel documentation systems (Grade GB004, Jencons PLC, London, UK) was applied for analysis of images.

Discussion
H. pylori is a common bacterium with high microbiological and clinical importance and about 50% of the world's population, depending to the geographic location considered, has been estimated to have been infected with this organism. Despite the high incidence of the infection, the reservoir for H. pylori and the routes of infection are still indeterminate and various routes of transmission have been recommended [23]. Moreover, epidemiological investigations suggest that transmission of H. pylori between individuals happens both via the oral-oral and fecal-oral routes [23]. In keeping with this, fecal-oral transmission has more significant implications than since H. pylori may occur in food and water supplies subsequent to fecal contamination [24]. Besides, the isolation of H. pylori in drinking water [13,14], raw vegetables [7,9], salads [7,9], meat [25,26], ready to eat foods [27,28], sterilized foods [29,30] and foods with animal origin such as milk [31][32][33][34][35], suggests that these foods may act as vehicles for transmission of H. pylori to human population.
The present study was performed to assess the prevalence rate, genotyping patterns and antibiotic resistance properties of H. pylori strains isolated from different types of raw milk samples. Totally, 10 [38] revealed that the prevalence of H. pylori in raw milk samples of different parts of Sudan had a range of 7 to 38%. Similar results have been reported for the high prevalence of H. pylori in milk samples from Japan (72.20%) [32], Greece (20.00%) [34], Italy 1.80%) [39] and Iran (16.00%) [40].
Foods presenting intrinsic factors, including water activity higher than 0.97 and pH ranging from 4.9 to 6.0 such as raw milk, theoretically could provide conditions for survival of H. pylori [7][8][9]. Therefore, it is not surprising that the H. pylori strains has the high prevalence in raw milk samples of our investigation. High prevalence rate of H. pylori in milk samples of our research is may be due to the low levels of hygienic conditions of milking procedure. Furthermore, considering the boost prevalence of H. pylori in healthy human carrier, contamination due to poor hygiene management of open package of milk, has more important implications for the transmission of the infection through foods. Milk, that could become contaminated during production or because of low hygiene after the open of package, is considered to be one of the most likely vehicles for infection [31,32]. Insufficient post-processing hygienic management of the milk, can carry the contamination of the matrix by humans. Despite of the low prevalence of H. pylori strains in some kinds of studied milk samples, the infectious dose of H. pylori is presumably low [31,32]. Therefore, it is an important public health threat regarding the consumption of raw milk. The urea-dependent acid resistance of H. pylori may account for the long-term survival of H. pylori in an acidic environment including raw milk [29]. Higher prevalence of H. pylori in raw ovine milk samples is may be due to the more suitable conditions present in ovine milk such as higher fat, protein and water activity and also optimum pH. Furthermore, ovine milk may have a higher qualification for growth and survival of H. pylori strains. Moreover, differences in the feed of ovine with bovine, buffalo, camel and even caprine species may affect the prevalence rate of bacteria presented in their milk. Higher prevalence of H. pylori in raw ovine milk was also reported by previous investigations [10,31,32,[34][35][36][37][38][39][40][41][42][43].  Using thorns and thistles in deserts and living away from humans and the polluted environment of cities are the most important probable reasons for the lower prevalence of H. pylori in camel milk. Lower prevalence of H. pylori in raw camel milk was also reported by previous investigations [10,37,44,45]. We described that H. pylori bacteria exhibited the maximum prevalence of resistance against ampicillin, tetracycline, amoxicillin, metronidazole and erythromycin antibiotics. Boost prevalence of resistance against human-based antibiotics such as metronidazole, erythromycin, clarithromycin, levofloxacin, amoxicillin, streptomycin, rifampin, cefsulodin, trimethoprim, furazolidone and spiramycin in H. pylori bacteria isolated from raw milk samples characterized their anthropogenic origin. Reversely, boost prevalence of resistance against animal-based antibiotics such as ampicillin and tetracycline in H. pylori bacteria isolated from raw milk samples characterized their animal origin. As it displayed, majority of H. pylori bacteria exhibited resistance against human-based antibiotics. Extreme, illegal and prohibited prescription of antibiotics in medicine and also veterinary caused momentous surge in antibiotic resistance. bacteria of human clinical specimens displayed boost prevalence of resistance against aminoglycosides, tetracyclines, penicillins, macrolides and metronidazole [47] which was parallel to our results.
A possible relationship between virulence factors and antimicrobial resistance has been suggested. A study conducted in 2009 in Ireland reported that the absence of cagA may be a risk factor for developing metronidazole resistance [57]. Other studies have found an association between clarithromycin resistance mutations and the less virulent vacA genotypes [58]. Another report revealed that cagE and vacA S1 correlated with clarithromycin and metronidazole resistance [59], while others found that neither cagA nor vacA was associated with resistance [60,61]. Therefore, it is important to found any significant relationship between the presence of virulence markers and antibiotic resistance amongst the H. pylori strains.
Triple therapy, including two antibiotics, amoxicillin and clarithromycin, and a proton pump inhibitor given for a week has been recommended as the treatment of choice at several consensus conferences [62]. However, this treatment may fail for several reasons, as reported elsewhere [63]. In fact, the main reason for failure was found to be H pylori resistance to one of the antibiotics used (that is, clarithromycin). Other treatments have also been proposed, including metronidazole, a drug for which resistance is also a problem although to a lesser extent, as well as tetracycline, fluoroquinolones, and rifamycins for which resistance has become an emerging issue [64]. Results of the present investigation showed that application of furazolidone, streptomycin and cefsulodin may be effective for treatment of the cases of H. pylori infections. Reduction in the antibiotic prescription and also prescription of antibiotics according to the results of the disk diffusion can reduce the risk of antibiotic resistance. Using medicinal plants and especially those with high antimicrobial effects is a practical alternative way for treatment of H. pylori infection.

Conclusions
To put it in a nutshell, we recognized a great numbers of virulent and resistant H. pylori bacteria in raw milk samples of bovine, ovine, caprine, buffalo and camel species. Boost incidence of H. pylori bacteria in raw milk characterizes that these samples may be the natural reservoirs of the bacteria and can spread H. pylori to human. Moreover, some of the H. pylori bacteria of our research harbored vacA, cagA, iceA, oipA and babA2 genotypes together which represents the high pathogenicity. Furthermore, higher prevalence of iceA1+ strains than iceA2+, oipAthan oipA+ and finally babA2than babA2+ is another important finding of our study. Additionally, presence of 97 diverse combined genotyping patterns with high distribution of s1a/cagA+/iceA1/ oipA−/babA2-, m1a/cagA+/iceA1/oipA−/babA2-, s2/ cagA+/iceA1/oipA−/babA2-, s1a/cagA+/iceA1/oipA −/babA2+, m1a/cagA+/iceA1/oipA−/babA2+, s2/cagA +/iceA1/oipA−/babA2+, s1a/cagA+/iceA1/oipA+/babA2and m2/cagA+/iceA1/oipA−/babA2+ is another interesting finding of our research. Similarities in the genotyping pattern of H. pylori strains between various milk sources represent their same route of infection. High prevalence of multi-drug resistant H. pylori strains shows that raw milk of bovine, ovine, caprine, buffalo and camel species may be reservoir of antibiotic resistant H. pylori. Prescription of cefsulodin, furazolidone, spiramycin and streptomycin may be effectual for treatment of cases of H. pylori infections due to the consumption of raw milk. Additional researches are essential to recognize the rates of the molecular genetic homology of H. pylori bacteria isolated from milk and dairy samples and those of human clinical specimens to confirm the zoonotic aspects of H. pylori.