Isolation and identification of Helicobacter pylori from raw chicken meat in Dhamar Governorate, Yemen.

Although Helicobacter pylori (H. pylori) is one of the most common bacterial pathogens of human, its natural reservoirs are still unclear. There is an increasing number of reports that document the occurrence of H. pylori in various foods. This study aimed at isolation of H. pylori from chicken meat sampled. Two hundred and sixty samples were collected randomly from slaughterhouses and markets in Dhamar Governorate, Yemen. Samples were enriched in Brain-Heart Infusion broth in microaerophilic conditions before inoculating the Camp-Blood agar and EYE agar plates. Results showed that 13.8% of samples were contaminated evidenced by H. pylori growth via traditional culture method on agar media. No significant differences between sample types (thighs and breast muscles) (p=0.353) or the sampling source (p=0.816) were observed. Autumn season was associated with increased occurrence of H. pylori. The source of H. pylori in food is still not identified. Proper cooking and good sanitation practices are highly recommended to avoid the infection. Further studies addressing the potential sources of H. pylori are highly suggested. ©Copyright: the Author(s).

Introduction

Helicobacter pylori (H. pylori) is one of the most prevalent human bacterial pathogens globally (Sjomina et al., 2018). It is estimated that about two-thirds of the world’s population are infected with H. pylori, predominantly in developing countries with higher occurrence in poor and unhygienic areas. The prevalence of H. pylori infection depends on diverse contributing factors such as socioeconomic status, geographical area, living conditions, and personal hygiene (Sjomina et al., 2018; Al Mashhadany, 2020). Infected individuals are the main reservoir of H. pylori, however, most of these infections are asymptomatic (Denic et al., 2020). Clinically, H. pylori infection in human is associated with chronic gastritis, peptic ulceration, duodenal ulcer, gastric cancer as well as mucosa associated lymphoid malignancies (Almashhadany & Mayass, 2018; Denic et al., 2020).

From bacteriology perspective, H. pylori is ~2-3.5×0.5-1.0 μm small, curved, microaerophilic, lophotrichous gram-negative, S-shaped or curved rod bacterium. It has copious amounts of urease enzyme to survive the acidic environment of the stomach by converting urea to ammonia. The production of ammonia around H. pylori neutralizes the acidity of the stomach, making it more hospitable for H. pylori. Moreover, the helical shape of H. pylori allows it to be hidden in the mucus layer which is less acidic than the surface or the lumen of the stomach (Saeidi & Sheikhshahrokh, 2016; Al-Mashhadany et al., 2018).

Molecular epidemiology studies had detected H. pylori DNA in different foodstuffs, water, and animals which suggest the existence of reservoirs for H. pylori outside human gastrointestinal tract (Momtaz et al., 2014; Mousavi et al., 2015). Milk, meat, and vegetables are a potential source of H. pylori infections (Duynhoven & Jonge, 2001; Herrera, 2004). Milk products are the most studied, probably because the infection is mainly acquired during childhood and milk is mostly consumed during this period (Al-Mashhadany & Mayass, 2017; Talimkhani & Mashak, 2017). Nonetheless, the role of foods as a transmission medium is not well-validated clinically. The most commonly accepted hypothesis of H. pylori transmission is the oral-fecal route (Sjomina et al., 2018). Despite the absence of solid evidence of foods as a reservoirs for H. pylori, different studies had isolated or identified different strains and raised concerns about the contribution of nonhuman sources (Keenan et al., 2010; Talimkhani & Mashak, 2017; Hamada et al. 2018). Suboptimal sanitation conditions are favored for oral-fecal & oral-oral transmission of H. pylori in institutions of disable individuals and orphanages (Sjomina et al., 2018). Several studies have reported the survival and presence of H. pylori in foods and water, particularly in ready-to-eat products and milk, proposing that they can be sources of infection (Quaglia & Dambrosio, 2018). Foods intrinsic factors, such as pH ranging (4.9 to 6.0) and water activity (>0.97) could theoretically provide good conditions for H. pylori survival. Therefore, data on survival ability may be more significant than concerns about the growth of the bacteria in foods when determining the role of different types of food in H. pylori transmission to humans (Quaglia et al., 2007; Quaglia & Dambrosio, 2018; Al-mashhadany 2018). The prevalence of H. pylori infection in Yemen is not well-defined as various studies reported a wide range of 10-82.2% (Gunaid et al. 2003, Al-Shamahy 2005, Bahumid et al. 2009, Almashhadany & Mayass 2018), and its transmission routes in poor developing countries are a matter for wide debated (Alsulaimany et al. 2020). This study was conducted to detect the occurrence of H. pylori in raw chicken meat at Dhamar Governorate (Yemen). The relationship between occurrence of H. pylori in chicken meat and months during the period of study was also addressed.

Materials and Methods

Study design and sampling

Two hundred and sixty (260) fresh raw chicken meat samples (140 Thigh, and 120 Breast), were collected from retail markets and chicken slaughterhouses in different places of Dhamar Governorate, from July to December 2020. The samples were put in sterile cooled polyethylene bags and kept in ice box with temperature approximately 4°C during transport and storage at the laboratory (Almashhadany, 2021b). The bacteriological analysis was performed within 2 h of sample collection.

Isolation of H. pylori

In the laboratory, the isolation of H. pylori was done under aseptic conditions as previously published (Al-Mashhadany & Mayass, 2017; Almashhadany, 2021a). Briefly, samples were cut into small pieces using sterile blades for liberation of adherent bacteria. From thigh and breast, 25 gm (as an optimal sample size) was soaked in 250 ml of normal saline. For enrichment, a volume of 0.5 ml of the suspension was then placed in a 4.5-ml Brain-Heart Infusion broth with 7% horse serum without antibiotics and incubated in a microaerophilic atmosphere (GasPack; Oxoid, Basingstoke, England) at 37°C for 3 to 7 days. After that, modified Campyblood agar and EYE agar plates were inoculated with 100 μl of the enriched suspension and incubated at 37°C in microaerobic condition in a candle jar and Campy Gen (2.5 L) in the incubator for 4-10 days. For purification purposes, developed colonies were subcultures on the same agar media and incubated at 37°C for 48–72 hrs. (Coldham et al., 2011; Lawson, 2015).

Identification of H. pylori

Identification of H pylori isolates was done according to a published standard scheme (Lawson, 2015; Al-Mashhadany & Mayass, 2017). Briefly, after incubation, all cultural plates were examined for suspected colonies of H. pylori. Gram staining was done according to the standard protocol with exposure of smears to safranin for 3 minutes. Biochemical tests employed for the identification included: Catalase, Oxidase, Urease, Indole production, growth in 1% glycine, growth in 3.5% NaCl, H2S production in (TSI), TSI with lead acetate paper, resistance to nalidixic acid, sensitivity to cephalothin, and hippurate hydrolysis. Isolates that met the reference characteristics were considered H. pylori (Lawson, 2015; Al-Mashhadany & Mayass, 2017).

Statistical analysis

Data were analyzed using SPSS software (version 25), confidence intervals (CI) were estimated using normal distribution approximation at an alpha level of 0.05. Chi-square test was used to evaluate differences between groups.

Results

Occurrence of H. pylori in raw chicken meat samples

From 260 raw chicken meat samples, 18 (13.8%) showed a positive result for H. pylori. This result includes 11 (15.7 %) positive samples from thigh and 7 (11.7%) positive samples from breast (Table 1). There is no significant difference between sample types in terms of contamination with H. pylori (p=0.353). Based on this sample size, up to 18% of chicken meat samples are expected to be contaminated with H. pylori.

Table 1.

Occurrence of H. pylori in raw chicken meat according to type of meat.

Chicken meat	No. samples	Positive Samples n (%)	95% CI
Thigh	140	22 (15.7)	9.69 – 21.74
Breast	120	14 (11.7)	5.92 – 17.41
Total	260	36 (13.8)	9.65 – 18.04

Occurrence of H. pylori according to sampling location

Regarding to the distribution of H. pylori among examined samples, the results showed a slightly higher occurrence of H. pylori in samples from slaughterhouses (Table 2). However, this increase was not significant statistically (χ²=0.054, p=0.816).

Table 2.

Occurrence of H. pylori in raw chicken meat according to sampling location.

Chicken meat	Slaughterhouses		Retail markets
	No. of tested	Positive samples n (%)	No. of tested	Positive samples n (%)
Thigh	80	12 (15.0)	70	10 (14.3)
Breast	60	8 (13.3)	50	6 (12.0)
Total	140	20 (14.3)	120	16 (13.3)

Temporal distribution

The changes in occurrence of H. pylori were monitored throughout the study period. The highest rate of H. pylori was observed in October (23.8%) and September (22.7%), while the lowest rate was found in June (5.0%) and August (8.3%) (Figure 1). Autumn was significantly associated with increase in H. pylori contamination of chicken meat (p=0.007).

Figure 1.

Seasonal variations in occurrence of H. pylori during the period of study.

Discussion

The occurrence and survival of H. pylori in different foods have been a hot area of research during the past decades. Studies addressing the occurrence of H. pylori in meat are rare, whereas the majority of published literature focused on milk and milk products (Herrera 2004, Quaglia & Dambrosio 2018). Recently, stomach of domestic animals have been found to harbor high numbers of H. pylori, which suggests domestic animals as an important reservoir (Saeidi & Sheikhshahrokh 2016). Therefore, this study aimed to detect H. pylori in chicken raw meat

Out of two hundred and sixty (260) raw chicken meat samples collected in this study, 36 (13.8%) were contaminated with H. pylori. This result is consistent with studies from Iran that found 10-14% of salad and vegetable samples harbored H. pylori (Atapoor et al. 2014, Yahaghi et al. 2014). Hemmatinezhad and associates in Iran, reported that 13.45% of ready-to-eat food samples were contaminated with H. pylori (Hemmatinezhad et al. 2016). Likewise, a similar occurrence was reported in raw milk detected by bacteriological culture or by molecular detection of ureC gene (Rahimi & Kheirabadi 2012, Kazemeini et al. 2014, Talaei et al. 2015). On the contrary, other studies documented higher rates (20-36%) in different foods including chicken raw meat (Dore et al. 2001, Meng et al. 2008, Mousavi et al. 2015, Saeidi & Sheikhshahrokh 2016). The role of food prepared under poor hygienic conditions as a possible vehicle for H. pylori transmission was suggested by Begue and colleagues, who found significant hazards for consumption of food obtained from street vendors in Peru (Begue et al. 1998). The actual sources of H. pylori in chicken raw meat have not been identified. However, contaminated water, infected handlers, and chicken gastrointestinal tract are the most probable sources (Meng et al. 2008, Vale & Vítor 2010, Quaglia & Dambrosio 2018).

Regarding the distribution of H. pylori among examined samples, it seems that sample type is not a contributing factor for occurrence of H. pylori. This observation is supported by a recent study in poultry slaughterhouses in Egypt that found 3.33% of liver samples to be contaminated with H. pylori, while 2.22% samples of meat and gizzard were contaminated (Hamada et al. 2018). According to our findings and previous investigations, H. pylori truly occur in foods (Duynhoven & Jonge 2001, Atapoor et al. 2014, Saeidi & Sheikhshahrokh 2016, Quaglia & Dambrosio 2018). However, techniques for direct isolation of H. pylori from foodstuff have not been fully developed or standardized. Indeed, the isolation of H. pylori from food products is quite difficult due to the presence of associated microflora and to the probably very low H. pylori load (Vale & Vítor 2010, Talimkhani & Mashak 2017).

The relationship between months and occurrence of H. pylori during the work in Dhamar Governorate was studied. The highest rates of isolation of H. pylori were found in October (23.8 %) and September (22.7 %). However, the occurrence of H. pylori was seen to decrease prior September and after October. This observation contradicts the previous study that did not find H. pylori in February, March, July, August, and September (Al-Mashhadany & Mayass 2017). In fact, the seasonality of H. pylori occurrence in poultry meat is still unaddressed. Autumn is a wet season in Dhamar that may provide favorable conditions for H. pylori proliferation in animals’ gastrointestinal tracts that is reflected by higher contamination observed in September and October.

Conclusions

H. pylori occurs in foods and may be an important media for its transmission (horizontal method). The occurrence of H. pylori in raw chicken meat in Dhamar Governorate seems to be high, mostly due to poor living conditions, socioeconomic status, and sanitary habits, or other risk factors. The seasonality of H. pylori in poultry meats is still unclear. In addition, special emphasis on proper cooking of chicken meat before consumption is recommended.