OCCURRENCE OF HELICOBACTER SPECIES WITH SPECIAL PRIORITY TO H. PYLORI IN HEN'S EGGS

Assiut University web-site: www.aun.edu.eg

OCCURRENCE OF HELICOBACTER SPECIES WITH SPECIAL PRIORITY TO

H. PYLORI IN HEN'S EGGS

MARWA EZZAT ABDEL GAID KHOLEAF¹, MOSTAFA KHALIL MOSTAFA²,

NAGAH MOHAMMED SAAD² and MARWA MOHAMMED NABIL HASSAN¹

¹ Department of Food Hygiene, Animal Health Research Institute, Assiut Lab.

² Department of Food Hygiene, Faculty of Vet. Med., Assiut University, Egypt

Received: 24 June 2018;     Accepted: 9 July 2018

INTRODUCTION

Eggs are among the few foods that are used throughout the world regardless of religion and ethnic groups (Stadelman and Cotterill, 2001). The hen’s egg is one of the perfectly preserved biological items found in nature and its nutritional benefits are well recognized. Eggs remain a stable food within the human diet, consumed by people throughout the world in various dishes and considered very nutritious and a cheap source of protein (Osei-Somuah et al., 2003).

Eggs also have many functional properties such as foaming, emulsifying and unique color and flavor, which are important in several food products (Stadelman and Cotterill, 2001). Freshly laid eggs are generally devoid of organisms, however, following exposure  to  environmental  conditions,  may become

Corresponding author: Dr. Marwa Mohammed Nabil Hassan

E-mail address: ahmednofel122@yahoo.com

Present address: Department of Food Hygiene, Animal Health Research Institute, Assiut Lab.

contaminated with different types of microorganisms (Ellen et al., 2000). Eggs are liable to contamination either before laying (congenitally) or after laying (extragenitally), when the microorganisms reach the egg contents through penetration pores of shell cause low egg quality, low shelf life, low safety and induce public health hazards (Board and Fuller, 1994). In addition, fecal matter, improper washing, using of contaminated water and bad handling are the common sources of contamination. In spite of their high nutritive value, eggs were responsible for several outbreaks and were a vehicle for transmission of certain human pathogens as Helicobacters (Miyamota et al., 1998; Hangombe et al., 1999; Gast et al., 2004).

Helicobacter is a genus of Gram-negative bacteria possessing a characteristic helical shape. They were initially considered to be members of the Campylobacter genus, but in 1989 Goodwin et al. published sufficient reasons to justify his new genus name of Helicobacter. The Helicobacter genus contains about 35 species and some species are pathogenic and known to colonize the gastrointestinal and biliary tracts of many animal species. They are grouped into two groups gastric and enterohepatic Helicobacter species(Boyanova, 2011).

Gastric Helicobacter species including H. pylori,H. acinonychis,H. bizzozeronii,H. felis,H. mustelae,H. nemestrinae,H. salomonisandH. suncus. Enterohepatic Helicobacter species as H. pullorum, H. bilis,H. canadensis, H. canis,H. cholecystus,H. cinaedi, H. fennelliae, H. ganmani, H. hepaticus, H. mesocricetorum,H. muridarum, H. pametensis,H. rodentium,H. trogontumandH. typhlonicus)Solnick and Vandamme, 2001).

There are many Helicobacter species that infect human and leading to many medical condition, as H. helimannii which leads to duodenal ulcer, gastric carcinoma and mucosa associated lymphoid tissue (MALT) tumors. Also H. felis which is usually associated with gastric disease and many species isolated from human diarrheal samples included H. cinaedi, H. canis, H. pullorum, H. fennellae, H. canadensis and H. pylori. Additially, there are Helicobacter species have been isolated from livers and have been associated with hepatic diseases as H. hepaticus which lead to hepatitis and hepatic carcinoma(On et al., 2002).

The best known and the most important in terms of global impact on human disease is H. pylori in which the definitive reservoir is assumed to be humans(Drumm et al., 1990).The history ofdiscovering and isolation of H. pylori was first documented since a century ago. H. pylori was first discovered in the stomachs of patients with gastritis and ulcers in 1982 by (Marshall and Warren 1983)and they awarded the 2005 Nobel Prize in Physiology or Medicine. Acute infection with H. pylori may appear as an acute gastritis with abdominal pain or nausea(Butcher, 2003) which develop to chronic gastritis. The symptoms are often stomach pains, nausea, bloating, belching, and sometimes vomiting or black stool(Ryan, 2010).

About 10-20% of those infected with H. pylori develop gastric and duodenal ulcers. H. pylori infection is also associated with 1-2% lifetime risk of stomachcancer and less than 1% risk of gastric mucosa associated lymphoid tissue lymphoma (MALT)(Kusters et al., 2006).H. pylori has been recognized as the principal cause of gastric disease which is more severe than that caused by H. helimannii and cause gastritis, peptic ulcer, gastric carcinomas, and mucosa- associated lymphoid tissue lymphoma (Komoto et al., 1998). H. pylori was classified as a definitive carcinogen to human which may play a causative role in development of up to 90% of gastric cancers (Forman et al., 1994).

There was association between H. pylori infection and hypertension and myocardial infarction. (Whincup et al., 1996). H. pylori infection presents approximately in half of the world's population (Lambert et al., 1995).The organism can be found in 70-90% and 25-50% of the population in developing and developed countries, respectively(Sykora et al., 2006; Vale and Vitor, 2010) depending on environmental and socioeconomic factors.  In spite of the general idea about the low prevalence of gastric cancer, H. pylori infection considered the fourth most common type of cancer and the second leading cause of cancer- related deaths worldwide(WHO, 2010). The minimum infectious dose of H. pylori during primary and secondary infection was 10⁴ bacteria (Solnick et al., 2001).

Moreover, Enterohepatic Helicobacter species, including H. pullorum, is increasingly recognized as microbial pathogen in humans and animals (On et al., 1996; On et al., 2002). Helicobacter pullorum was first described by Stanley et al. (1994). On et al. (1996)indicated that H. pullorum was originally isolated from the feces, the intestines and damaged livers of broilers and laying hens (Burnens et al., 1994; Stanley et al., 1994), and also from the feces of humans (Ceelen et al., 2005). H. pullorum has been related to enteritis and hepatitis in broiler chickens and laying hens, diarrhea, gastroenteritis, and liver disease in humans (Burnens et al., 1994; Stanley et al., 1994; Young et al., 2000 and Ceelen et al., 2005). The organism can be considered a food borne human pathogen (Ceelen et al., 2006).

Fox et al. (1998)observed that H. pullorum, H. bilis and H. rappini were found to cause some kinds of extra digestive diseases in liver and gall bladder of human with chronic cholecystitis.

Since the isolation of Helicobacter speciesfrom eggs is very scarce and to determine whether eggs act as a vehicle in transmission of Helicobacterspecies, therefore this work was planned to study the incidence of Helicobacter species in hen's eggs, in addition to identification and confirmation of H. pylori ure C gene by using PCR.

MATERIALS and METHODS

Isolation and identification of Helicobacter species from hen's eggs.

Collection of samples:

A total of 300 random eggs, representing 60 samples, (30 from baladi hens', and 30 from poultry farms) were collected from, poultry farms, groceries and supermarkets located in Assiut Governorate, Egypt. Each egg sample (composed of 5 eggs) was placed in a sterile plastic bag and dispatched to the laboratory with a minimum of delay where they were prepared and examined.

Preparation of samples:

Egg shells: Egg shells were tested by a surface rinse method as described by Moats (1980).

Egg contents: The egg sample was prepared for evacuation of its content according to Speck (1976).

Isolation of Helicobacter species: The technique adopted by Stevenson et al. (2000) was used.

Enrichment: One milliliter of rinsed solution and egg content samples were aseptically inoculated into a sterile test tubes containing 10 ml of selective enrichment broth Helicobacter pylori special peptone broth (HPSPB) containing selective supplement which is Vancomycin5.0 mg, Trimethoprim lactate 2.5 mg, Cefsulodin 2.5 mg and Amphotericin B 2.5 mg. The inoculated tubes were incubated at 37^oC for 48 hours in an atmosphere of 6% O₂, 10% CO₂ and 84% N₂ by CO₂ incubator (HERA cell 150 Thermo scientific).

Selective plating: Loopfulls from incubated broth cultures were then streaked on HPSPA supplemented with Helicobacter selective supplement (Oxoid Code SR147E).All the cultured plates were inspected after 3, 5 days. Suspected colonies grow, slowly, small not exceeding 2 mm in diameter, translucent, circular and convex.

Identification of Helicobacter (Solnic and Vandamme, 2001):Gram stain and Biochemical characteristicswhich include Oxidase production test, Catalase production test, Urease production test, Hippurate hydrolysis test, Nitrate reduction test, Glycine tolerance test and Salt tolerance test (Solnic and Vandamme, 2001).

Identification of ureC gene for H. pylori by using PCR.

DNA Extraction using QIA amp kit (Shah et al., 2009):

After overnight culture on nutrient agar plates, one or two colonies were suspended in 20 ml of sterile distilled water, and the suspension was then heated at 100ºC for 20 minutes. Accurately, 50-200 µl of the culture were placed in Eppendorf tube and Equal volume from the lysate (50-200 µl) was added, after addition of 20-50µl of proteinase K, the mixture was incubated at 56 ºC for 20-30 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The solution was added to the column and centrifuged at 8000 rpm for 1 min, then the filtrate was discarded. The sediment was washed using AW1 buffer (200 µl), the column was centrifuged at 8000 rpm / 1 min, and the filtrate was discarded. Washing was applied by using the AW2 buffer (200µl), the column was centrifuged at 8000 rpm / 1 min, and the filtrate was discarded. The column was placed in a new clean tube then, 25-50 µl from the Elution buffer was added, centrifuged at 8000 rpm/1min, and then the column was discarded. The filtrate was put in clean tube containing the pure genomic DNA and nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

Amplification reactions of DNA:

Amplification of ureC gene for H. pylori (Kianpour et al., 2014):

The amplification was performed on a Thermal Cycler (Master cycler, Eppendorf, Hamburg, Germany) using 50 µL containing 5 µL 10 × 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 conditions: an initial denaturation for 10 minutes at 94°C; 35 cycles for 1 minute at 94°C, 1 minute at 55°C, 1 minute at 72°C, and a final extension at 72°C for 10 minutes.

PCR amplified products were analyzed by 1.5% of agarose gel electrophoresis in 1x TBE buffer stained with ethidium bromide and visualized on UV transilluminator. A 100 bp plus DNA Ladder was used to determine the fragment sizes and the gene product was 294 bp.

Primer sequences used for PCR identification system:

Application of PCR for detection of urease C gene (ureC) aka phosphoglucosamine mutase gene (glmM) specific for identification and characterization of H. pylori was performed essentially by using Primers (Pharmacia Biotech) as shown in the following table:

RESULTS

Table 1: Incidence of isolated Helicobacter species in the examined hen's eggs shell samples.

Table 2: Incidence of different isolated Helicobacter species recovered from the examined hen's eggs shell samples.

Table 3: Incidence of isolated Helicobacter species in the examined hen's eggs content samples.

Table 4: Incidence of different isolated Helicobacter species recovered from the examined hen's eggs content samples.

Table 5: Comparison between the incidence of H. pylori in the examined hen's egg shell samples by using biochemical and PCR technique.                                                                                                  

Table 6: Relation between biochemical and PCR technique in isolated H. pylori.  

Photograph 1: Agarose gel electrophoresis of PCR amplification products using ureC gene as specificprimer for identification of H. pylori.

Lane M: 100 bp ladder as molecular DNA marker.

Lane 1: Control positive H. pylori for ureC gene.

Lane 11: Control negative for H. pylori.

Lanes (2, 3, 4, 6, 7, 8, 9 and 10): Positive strains as H. pylori.

Lane 5: Negative H. pylori isolated frombalady egg shell samples.

Lanes (2, 3, 6, and 9): Strains isolated from balady egg shell samples.

Lanes (4, 7, 8 and 10): Isolated strains from poultry farms egg shell samples.

DISCUSSION

The idea of the present study dealing with the incidence of Helicobacter species in hen's eggs was coming from the fact that the organism was originally isolated from the feces of broilers and laying hens (Burnens et al., 1994, Stanley et al., 1994). In addition, a preliminary study showed its presence on 60% of poultry carcasses pointing to it as a potentially important food- associated human pathogen.

Therefore, the discovery of Helicobacter species has sparked an interest in exploring the pathogenic potential of these organisms in food especially in eggs because eggs are considered as an essential food element for growth and maintenance of human health in addition to its high nutrient contents, low caloric value and ease of digestibility. However the nutrients that make eggs a high – quality food for human are also a good medium for bacteria (Frazier and Westhoff, 1986).

It was observed from Table 1 that shells contamination with Helicobacter species on (HPSPA) in balady hens eggs was (33.33%) and in poultry farms eggs was (30%). This confirms the opinion of Burnens et al. (1994) and Stanley et al. (1994) that the contamination may came from feces of hens. The relatively high incidence of Helicobacter species contamination was observed in examined balady hen’s eggs shell samples in percentage of (33.33%), and in percentage of (30% ) in the examined egg shell samples from poultry farms hen’s eggs. This is could be attributed to the bad hygiene during the production of eggs. Also, eggs are liable to contamination from feces of hens according to (Burnens et al., 1994, Stanley et al., 1994) who stated that the organism was originally isolated from the feces of broilers and laying hens. The Helicobacter species are recovered from the examined balady hen's egg content indicating the bad hygienic measures in the farmer's houses.

Data summarized inTable 1indicated that the contamination of the balady hen's eggs shell samples was slightly higher than the contamination of the poultry farms egg shell samples. Presence of Helicobacter species in both types of egg indicate the bad hygienic measures of the production of hen's eggs. Data showed in Table 1 was less than the result obtained by Abdel Hameed and Sender (2011) indicating the good hygienic measures during production of eggs.

The external shell contamination could be important for the shell life and the food safety of consumption of eggs and egg’s products (Smith et al., 2000). This is observed from the obtained results in Table 2 that the identified Helicobacter species from examined balady hen's egg shells samples were H. pylori 5 (16.67%),  H.cinaedi 2 (6.67%), H. felis 2 (6.67%) and H. pullorum 1 (3.3