Mubarak et al.

BMC Veterinary Research (2023) 19:250 BMC Veterinary Research

https://doi.org/10.1186/s12917-023-03819-6

RESEARCH Open Access

Molecular characterization of Helicobacter

pylori isolated from Nile Tilapia (Oreochromis
niloticus) and fish handlers
Asmaa Gaber Mubarak1* , Hanan H. Abd-Elhafeez2* and Hams M. A. Mohamed3*

Abstract
Background Helicobacter pylori is a worldwide pathogen that affects both animals and humans with a wide
environmental distribution, causing serious health problems in humans. This research has timely addressed the
topic of new sources of H. pylori infection, which is currently a global issue, especially in developing countries. For
this purpose, 115 Tilapia fish, 50 freshwater samples, and 88 fish-handlers’ stool samples were investigated for the
presence of H. pylori in Qena Governorate, Egypt. The applied techniques were antigen screening tests, culturing, and
molecular methods through ureC gene amplification, and 16 S rRNA characterization.
Results Helicobacter pylori was detected in 7.83%, 14%, 4.35%, and 12% of the investigated fish and water samples
by culture and PCR methods, respectively. Out of the total studied participants, 40 tested positive for H. pylori
when screened by stool antigen test, of which 35 (39.77%), and 31 (35.23%) were confirmed by conventional and
molecular techniques, respectively. The Fisher’s exact test has shown a statistically significant correlation between H.
pylori infection, sex, and age as risk factors, while the association was insignificant concerning the residence. Males
contracted the infection at a higher rate than females (48.08% and 16.67%, respectively). Also, H. pylori infection rate
was the highest among fish-handlers aged 36–45 years old (46.67%), followed by the 26–35 years old age group
(39.53%). With regard to the residence, a higher occurrence rate was recorded in the rural (36.07%) than the urban
population (33.33%). Helicobacter pylori isolates harbored the highest antimicrobial resistance against ampicillin
(100%), metronidazole (95.24%), while the least antimicrobial resistance was recorded against levofloxacin (21.43%),
and clarithromycin (26.20%). The phylogenetic analysis revealed a high degree of homology between the isolates
selected from Tilapia fish, freshwater, and fish-handlers.
Conclusions Our data emphasized the role that fish and freshwater play in disseminating H. pylori infection as one of
the diseases that has a significant public health issue.
Keywords Antibiotic resistance, Fish, H. Pylori, Human, Phylogeny

1
*Correspondence: Department of Zoonoses, Faculty of Veterinary Medicine, South Valley
Asmaa Gaber Mubarak University, Qena 83523, Egypt
2
a_mubarek@vet.svu.edu.eg Department of Cell and Tissues, Faculty of Vet. Medicine, Assiut
Hanan H. Abd-Elhafeez University, Assiut 71526, Egypt
3
hhnnzz91@aun.edu.eg Department of Microbiology, Faculty of Veterinary Medicine, South
Hams M. A. Mohamed Valley University, Qena 83523, Egypt
hams.mohamed@vet.svu.edu.eg

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Mubarak et al. BMC Veterinary Research (2023) 19:250 Page 2 of 11

Background The fecal-oral route of transmission of H. pylori has

Helicobacter pylori infection has a significant public more important implications than the oral-oral route,
health impact on human health in both developed and as it occurs in food and water supplies after fecal con-
developing countries, as it is classified by the WHO as a tamination. Several studies have proven H. pylori to be a
bacterial carcinogen. Additionally, it is commonly asso- food-borne pathogen because of its microbiological and
ciated with chronic infections, and peptic ulcers [1]. epidemiological characteristics. Many individuals harbor
Helicobacter pylori is a microaerophilic Gram negative this organism and never develop any clinical symptoms;
spiral shaped bacteria belonging to the family Helicobac- however, others may develop signs of nausea, vomiting,
teraceae that particularly colonize the human gastric epi- diarrhea, abdominal pain, and fever. In some cases, some
thelium and have been isolated from the gastrointestinal complications may occur, such as meningitis, abortion,
tract of many animals, causing one of the most common Guillan-Barré syndrome, Reiter’s syndrome, cancer, and
infections worldwide [2, 3]. even death [17].
The genus Helicobacter comprises over 35 species, As H. pylori is a fastidious, slow-growing bacterium,
of which H. pylori is the most important in terms of and as the exposure to different environmental condi-
human health [4]. The global prevalence of H. pylori is tions changes its characteristic from rod shape to coccoid
around 50%, and the infection with this microbe may form, many authors indicated that the culture method
be of greater importance in developing countries where is very difficult. While successful isolation and identi-
70–90% of the population is infected with H. pylori, com- fication of H. pylori can be affected by a number of fac-
pared to 25–50% in industrialized countries [5]. In Egypt, tors; including the clinical specimen quality, the time
H. pylori infection is estimated to be 60–80% by sev- between sampling and culture, and improper transport
eral studies [6, 7]. Helicobacter pylori infection has been conditions; nevertheless, the culture method is still con-
linked to the emergence of different gastro-intestinal dis- sidered the gold standard. In addition, H. pylori stool
eases, such as gastric ulcers and gastric cancer [8]. antigen test is rapid, accurate, and has a high sensitivity
As the bacterium can survive in animals’ stomachs, and specificity for detection of active and recent H. pylori
food products such as milk, meat, and vegetables are infection [18]. Moreover, the specificity and sensitivity
crucial in the spread of H. pylori infection [9]. On the of anti-H. pylori antibody test are 79–90%, and 76–84%,
other hand, water plays a critical role in the transmission respectively. Molecular methods are commonly used to
of the infection, either as an environmental reservoir or detect or confirm H. pylori to get more accurate diagnos-
through fecal-oral transmission, especially in unhygienic tic performance [19], but they have not been accepted for
conditions. The association between H. pylori antibodies routine testing.
and those produced during the infection with two known The antibiotic resistance of H. pylori represents a great
waterborne pathogens; hepatitis A virus, and giardia; has problem because it affects the success of eradication
been documented, so, the possibility of H. pylori to be therapy, and therefore, its treatment has proven to be
waterborne has been raised [10]. This bacterium can nat- incredibly challenging. Over the past two decades, there
urally live in aquatic habitats such as lakes, rivers, drink- has been an increased antibiotic resistance rate of H.
ing water supplies, and coastal marine ecosystems, from pylori all over the world [20, 21]. Therefore, H. pylori has
which sea food may become contaminated [11]. been identified by the WHO as one of the 20 pathogens
Fish is a highly nutritious food containing proteins, that represent the greatest harm to human health [17].
unsaturated fatty acids, minerals, and vitamins, so it’s The resistance of H. pylori to metronidazole, clarithro-
widely marketed and consumed [12]. Nevertheless, the mycin, levofloxacin, amoxicillin, and tetracycline has
slightly acidic pH of fish meat, besides its chemical com- been reported. While H. pylori antibiotic resistance var-
position, makes it highly susceptible to deterioration and ies regionally, there is no consistent report on whether it
contamination by different biological agents [13]. Inter- is related to age and gender [22]. Due to H. pylori being a
estingly, H. pylori was detected in Tilapia in numerous significant human pathogen, there is an ongoing need to
aquatic environments at various localities, unlike other identify additional environmental sources of this bacte-
fish species, even those taken from the same water source rium as potential routes for its dissemination and trans-
[14]. Nile Tilapia (Oreochromis niloticus) is among the mission to humans. Therefore, this study was designed to
most popular and cheap fish for Egyptians, and has the ascertain the presence of H. pylori in fish, natural envi-
ability to withstand in low oxygen, and high ammonia ronmental water, and humans. As well as, to describe
levels, so it can survive in poor environmental conditions some risk factors may related to human infection as sex,
[15], making it more susceptible to contamination by sev- age and residence. Furthermore the relationship between
eral biological contaminants; in particular bacteria; as H. the isolates and their antibiotic resistance.
pylori [14, 16].
Mubarak et al. BMC Veterinary Research (2023) 19:250 Page 3 of 11

Materials and methods Isolation and identification of H. pylori

Ethical declaration The seropositive stool samples, as well as fish intestinal
The Research Ethics Committee Regulations of South content, and water samples were subjected to isolation
Valley University, Qena, Egypt (VM/SVU/23(2)-28) were and identification of H. pylori according to Sainsus et al.
adhered in conducting this protocol. This study partici- [24], with slight modifications as follows: Aseptically, 10
pants provided their informed consent. All procedures mL of Brain Heart Infusion broth (BHIB) with a 5% Heli-
were carried out in compliance with the applicable rules cobacter selective supplement was added to a sterile test
and regulations. The study was conducted in accordance tube with 1 mL of each homogenized sample of human
with the ARRIVE (Animals in Research: Reporting In feces and fish intestinal content. A 0.22 μm Millipore
Vivo Experiments) criteria [23]. filter membrane was used to filter water samples. After
that, each membrane was submerged for 1 h in 2 mL of
Study design and sampling tryptic soy broth [25]. Then, 1 mL of TSB was diluted
Between February 2021 and November 2022, this with a 5% Helicobacter selective supplement, and added
study was conducted in Qena Governorate, Egypt, to the sterile test tube containing BHIB. With the aid of
located approximately 576 km from Cairo (26°10′12′′N, an active gas producing kit (5% H2, 5% CO2, 5% O2, and
32°43′38′′E). A total of 115 Tilapia fish were purchased 85% N2), inoculated test tubes were incubated at 37 °C in
from various markets and immediately transported to the a microaerophilic environment in an anaerobic jar and
laboratory for investigation in sterile ice boxes. For each monitored daily.
fish, the intestinal content was promptly obtained using For 96 h, the turbidity of the broth was tracked, and
a sterile scalpel blade and mixed with buffered peptone then it was streaked using the plating out method on
water (BPW) in sterile glass. Additionally, 50 water sam- Columbia agar with 5% defibrinated sheep blood sup-
ples were collected from different freshwater streams in plemented with Helicobacter selective supplement. The
the governorate (River Nile branches). In a sterile glass streaked plates were incubated at 37 °C for 3–5 days
(polypropylene), 250–500 ml of each sample was col- under microaerophilic conditions in an anaerobic jar as
lected, transported to the laboratory, and stored at 4 °C previously mentioned. The morphology of the suspected
for analysis. colonies, microscopic examination, and biochemical tests
In the same context, 88 fresh stool samples were col- (oxidase, catalase, urease, nitrate reduction, and hippu-
lected from fish handlers who exhibited symptoms of rate hydrolysis tests) were used to identify the colonies.
gastritis such as stomach pain, frequent burping, bloat-
ing, and unexplained weight loss. The samples were DNA extraction
collected in sterile, sealed cups, labeled, and promptly Extraction of H. pylori DNA from suspected colo-
transported to the laboratory in ice boxes for immedi- nies was done using QIAGEN QIAamp DNA mini kit
ate investigation. Sociodemographic characteristics of instructions.
the participants, including sex, age, and residence, were
documented. Molecular identification of H. pylori using the ureC (glmM)
gene
Antigen screening test According to Van Zwet et al. [26] (Table 1), the ureC
For the direct detection of H. pylori antigen in stool sam- (glmM) gene was the target of a PCR that was used to
ples from fish-handlers, the stool antigen test (SAT) is identify H. pylori. The PCR mixture contains;12.5 µL the
utilized which is a qualitative immunochromatographic Emerald Amp Max PCR Master Mix (Takara, Noji-higa-
method. Using an on-site H. pylori antigen quick test shiKusatsu, Japan), 1 µL of each primer, 4.5 µL of water,
cassette (CTK Biotech, USA), 50 mg of each sample was and 6 µL of DNA template. The amplification reaction’s
examined for the presence of H. pylori antigen following thermal profile included an initial denaturation at 95 °C
the manufacturer’s instructions. for 5 min, 30 cycles of 94 °C for 3 min, 94 °C for 1 min,
62 °C for 1 min, and 1 min at 72 °C, and a final extension
at 72 °C for 5 min. After electrophoresis, distinct bands at
417 bp were visualized.

Table 1 Primer sequences of Helicobacter pylori used in this study

Gene Sequence Amplicon size Reference
ureC gene EHC-V:(5′-CCCTCACGCCATCAGTCCCAAAAA-3′) 411 bp Van Zwet et al. [26]
EHC-L:(5′-AAGAAGTCAAAAACGCCCCAAAAC-3′)
16 S rRNA F27-AGAGTTT G ATC MTGGCTCAG 1500 bp Chèneby et al. [27]
R1492-TACGGTACC TTGTTACGACTT
Mubarak et al. BMC Veterinary Research (2023) 19:250 Page 4 of 11

Amplification and sequencing of the 16 S rRNA gene recommendations were followed while measuring zone
In order to partially amplify and sequence the 16SrRNA diameters.
gene, six isolates were randomly selected from freshwa-
ter, Tilapia fish, and fish handlers (two of each) (Table 1). Statistical analysis
The amplification condition and PCR components were SPSS version. 28 was used for the statistical analysis of
performed according to Chèneby et al. [27]. The PCR the study’s data. Through Geisser-Greenhouse’s epsilon
products were separated using 5 V/cm gradient elec- calculation, the connection between the positive H. pylori
trophoresis on 1.5% agarose gel (Applichem GmbH, infection and the sources of the investigated samples was
Darmstadt, Germany) at room temperature. 20µL of the determined. Fisher’s exact test was computed to establish
products were put into each gel slot. A gel documenta- the risk factors, and a 95% confidence interval (CI) was
tion system (Alpha Innotech, Biometra, Göttingen, Ger- calculated. For the variations in prevalence rates, p-val-
many) used to photograph the gel. The QIA quick PCR ues under 0.05 were regarded as significant.
Product extraction kit (Qiagen, Valencia, CA) was used
to purify the PCR products. For the sequence reaction, Results
Bigdye Terminator V3.1 cycle sequencing kit (Perkin- In this study, H. pylori was found to have a total incidence
Elmer) was used, and then was purified using Centrisep of 20.16% using conventional methods, and 16.60% using
spin column. DNA sequences were obtained by Applied PCR, as presented in Table 2; Fig. 1. As an unusual source
Biosystems 3130 genetic analyzer (HITACHI, Tokyo, of infection, the current study revealed a culture preva-
Japan). lence of H. pylori in Tilapia fish of 7.83% (9 out of 115)
collected from various freshwater streams. These iso-
Phylogenetic analysis lates displayed a negative nitrate reduction biochemical
Using the MegAlign module from the Lasergene pack- profile and positive results for catalase, oxidase, and urea
age (version 7), the phylogenetic tree was created. By hydrolysis tests. When confirmed using the ureC gene,
comparing our 16SrRNA sequences to those found in the only five samples (4.35%) tested positive. Out of the 50
database, MEGA6 software was used to create a neigh- freshwater samples, seven tested positive for H. pylori
bor-joining phylogenetic tree [28]. using conventional method, while six were positive using
PCR. Regarding the human stool samples, 40 of the 88
Phenotypic detection of antibiotic resistance fish-handlers initially tested positive for H. pylori through
According to Ranjabar et al. [29], antimicrobial suscep- an antigenic test, and their samples were subsequently
tibility testing for seven therapeutically useful antibiot- cultured, resulting in 35 positive samples (39.77%). How-
ics was completed using the Kirby-Bauer disc diffusion ever, when these isolates were further confirmed using
method on Muller-Hinton supplemented with 5% defi- the ureC gene, only 31 of them (35.23%) were confirmed
brinated sheep blood and incubated at 37 °C for 48 h in as H. pylori. No significant difference was found between
a microaerophilic atmosphere (85% N2, 10% CO2, and 5% human infection with H. pylori and the contamination
O2) in accordance with the Clinical Laboratory Standards levels of Tilapia fish and freshwater streams as potential
Institute’s (CLSI, [30]) recommendations. Ampicillin sources of infection (P = 0.2679).
(AM, 10), metronidazole (MET, 5), erythromycin (ER, 5), A total of 88 fish handlers agreed to participate as vol-
clarithromycin (CLR, 2), amoxicillin (AMX, 10), levoflox- unteers in this study, with 52 being male and 36 being
acin (LEV, 5), and rifampicin (R, 30) (Thermo Fisher Sci- female, as shown in Table 3. The incidence of H. pylori
entific, Basingstoke, United Kingdom) were evaluated for in human stool samples differed significantly (P = 0.0479)
their concentrations in micrograms per disk (mcg/disk). with 48.08% among males compared to 16.67% among
The Clinical Laboratory Standards Institute (CLSI, [30]) females. Participants’ ages ranged from 16 to over 45
years and were classified into four groups. Among these
age groups, participants aged between 36 and 45 years
Table 2 Helicobacter pylori in the examined samples had the highest H. pylori infection rate (46.67%), followed
The examined No. of Positive H. pylori Geisser- by the 26–35 age group (39.53%). These two age groups
samples examined Conventional PCR Green-
samples house’s
were significantly associated with the rate of H. pylori
methods
epsilon infection (95% CI, P = 0.0498). The age group of ˃ 45 years
No. (%) No. (%) R2 (P) exhibited an incidence of 30.77%, while, those aged 16–25
Tilapia fish 115 9 (7.83) 5 (4.35) 0.1175 had the lowest infection rate (17.65%). The residences of
Freshwater 50 7 (14) 6 (12) (0.2679) the participants were distributed as follows: 27 urban
streams and 61 rural. Rural residents had the highest prevalence
Fish-handlers 88 35 (39.77) 31 (35.23) of H. pylori (36.07%, 22/61) compared to urban residents
Total 253 51 (20.16) 42 (16.60) (33.33%, 9/27) with no significant difference (P ˃ 0.9999).
Mubarak et al. BMC Veterinary Research (2023) 19:250 Page 5 of 11

Fig. 1 Agarose gel electrophoresis of ureC (glmM) gene showing bands at 411 bp. Lane M: Marker (DNA ladder 100 bp), Lane P: positive control, Lane (1,
2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19): positive ureC (glmM) gene, Lane 9: negative isolate, and Lane N: negative control

Table 3 H. pylori isolated from humans in association with risk amoxicillin (76.19%, 32/42), while a low frequency
factors of resistance to levofloxacin (21.43%, 9/42), and clar-
Variable No. of Positive H. 95% CI ithromycin (26.20%, 11/42) was reported. As shown in
examined pylori P Table (4), the MAR index ranged from 0.571 to 1 in the
cases No. (%) obtainedH. pylori isolates, with the highest index value
Sex Male 52 25 (48.08) 0.0479*^ of 1 found in one isolate of fish-handlers’ stool. It’s worth
Female 36 6 (16.67) noting that most of H. pylori isolates were multi-drug
Age 16–25 y 17 3 (17.65) 0.0498* resistant (MDR) to at least three classes of antibiotics as
26–35 y 43 17 (39.53) observed in Tilapia fish and human isolates.
36–45 y 15 7 (46.67)
˃ 45 y 13 4 (30.77) Discussion
Residence Urban 27 9 (33.33) > 0.9999^
Helicobacter pylori has emerged as one of the most con-
Rural 61 22 (36.07)
cerning human pathogens associated with water, conse-
^ = Fisher’s Exact Test
quently, it has the potential to contaminate marine food
* Significant value
products. While few studies have addressed this issue, the
current study tried to evaluate the prevalence of H. pylori
The sequencing results of partially amplified 16SrRNA in fish, freshwater streams, and fish handlers, along with
for six isolates, selected randomly from fish intestinal an evaluation of some the risk factors associated with
contents (OQ456179, OQ456180), freshwater streams the infection. Culturing of Tilapia fish samples revealed
(OR144424, OR144425), and fish-handlers’ stool an incidence of H. pylori at 7.83%, and after molecular
(OQ456181, OQ456182), confirmed that our isolates confirmation through the use of the ureC gene, only five
were indeed H. pylori. The phylogenetic analysis revealed isolates (4.35%) were identified as H. pylori. Wang et al.
a high level of identity (99–100%) among our isolates, [31] found that although bacteriological culture is con-
despite their diverse sources. Additionally, our isolates sidered as the gold standard for infection diagnosis, H.
were grouped with reference strains from Genbank such pylori poses particular challenges. It requires additional
as H. pylori NR044761, H. pylori OP810348, and H. pylori resources for growth, a microaerophilic environment,
KC9009952 with identities of 97.3–97.4%, 100%, and and extended incubation, making it difficult to conduct
99.5–100%, respectively (Fig. 2). timely sensitivity studies. The technical difficulties asso-
Of the 42 H. pylori isolates tested for antibiotic resis- ciated with H. pylori culture are evident, highlighting
tance, the results are provided in Fig. (3). These results the need for simpler and quicker methods to identify H.
revealed complete resistance to ampicillin (100%), fol- pylori and assess its resistance. Molecular techniques
lowed by metronidazole (95.24%, 40/42), and rifam- have yielded promising results in this regard [32].
picin (88.10%, 37/42). Closely related resistance rates Many researchers have endorsed the use of the
were observed for erythromycin (78.57%, 33/42), and ureC (glmM) gene as a genetic marker for H. pylori
Mubarak et al. BMC Veterinary Research (2023) 19:250 Page 6 of 11

Table 4 Antibiotic resistance profile of H. pylori isolates chain. Moreover, Cortés-Sánchez [12] has proved that
Sources of No Antimicrobial resistance profile MAR Tilapia fish are a vehicle for the transmission of H. pylori.
isolates index
Helicobacter pylori is a significant emerging patho-
Tilapia fish 1 AM, MET, RIF, ER 0.571
gen associated with water, raising significant concerns
2 AM, RIF, AMX, CLR 0.571
3 AM, RIF, AMX, ER, CLR 0.714 for the scientific community [36]. Al-Sulami et al. [25]
4 AM, MET, RIF, ER 0.571 were the first to identify 10 isolates of H. pylori in water
5 AM, MET, AMX 0.429 through biochemical tests. Our study has confirmed the
Fresh water 1 AM, MET, AMX, ER 0.571 presence of 7 isolates from 50 freshwater samples (14%)
streams 2 AM, MET, RIF, ER 0.571 using cultural and biochemical methods. When con-
3 AM, MET, RIF, ER, CLR 0.714
4 AM, MET, AMX, ER 0.571
firmed by PCR, six isolates were obtained, reducing the
5 AM, MET, RIF, AMX, ER, CLR 0.857 rate to 12%. Twing et al. [37] were also able to detect
6 AM, MET, RIF, AMX, ER 0.714 H. pylori in freshwater samples. In Basrah Governor-
Human 1 AM, MET, RIF, AMX, ER, LEV 0.857 ate, Iraq, water samples have been contaminated by H.
diarrhea 2 AM, MET, AMX, CLR 0.571 pylori at an incidence of 2.76% [25]. Meanwhile, in Egypt,
3 AM, MET, AMX, ER 0.571 two isolates from tap water and ground water (one from
4 AM, MET, RIF, ER, LEV 0.714
5 AM, MET, RIF, AMX, ER 0.714
each) were identified as H. pylori through direct detec-
6 AM, MET, RIF, ER 0.571 tion of the ureC gene [38]. Other authors all over the
7 AM, MET, RIF, AMX, ER, CLR, LEV 1 world succeeded in detecting the organism in various
8 AM, MET, RIF, AMX 0.571 types of water using different techniques. Ranjbar et al.
9 AM, MET, RIF, ER 0.571 [29] revealed that 3% of drinking water samples had H.
10 AM, MET, RIF, AMX, ER 0.571
11 AM, MET, RIF, AMX,CLR, LEV 0.857
pylori through a combination of the culture method and
12 AM, MET, RIF, AMX, ER 0.714 the PCR technique based on 16 S rRNA. Also, H. pylori
13 AM, MET, RIF, AMX, LEV 0.714 could be detected in influent water samples in Colombia
14 AM, MET, RIF, AMX, ER, CLR 0.857 at a percentage of 22.6% [39]. Besides that, Farhadkhani
15 AM, MET, RIF, AMX, ER 0.714 et al. [11] detected H. pylori with a high frequency (36%)
16 AM, MET, RIF, ER, LEV 0.714
17 AM, MET, RIF, AMX 0.571
in wastewater samples, stressing that fecal pollution of
18 AM, MET, RIF, AMX, ER, CLR, LEV 1 water and other environmental samples plays an impor-
19 AM, MET, RIF, AMX 0.571 tant role in H. pylori infection transmission. On the other
20 AM, MET, RIF, AMX, ER 0.714 hand, Gholami-Borujeni et al. [1] reported that 24% of
21 AM, MET, RIF, AMX 0.571 water samples collected from urban areas, and 18% from
22 AM, MET, RIF, ER, LEV 0.714
23 AM, MET, RIF, AMX, ER 0.714
home water treatment devices in Iran tested positive for
24 AM, MET, RIF, ER 0.571 H. pylori.
25 AM, MET, RIF, AMX, ER, CLR 0.857 Differences in H. pylori prevalence in water could be
26 AM, MET, RIF, AMX,ER 0.714 attributed mainly to water characteristics including hard-
27 AM, MET, RIF, AMX, ER 0.714 ness, alkalinity, temperature, and pH, in addition to the
28 AM, MET, RIF, AMX, ER 0.714
29 AM, MET, RIF, AMX, ER, LEV 0.857
use of various methods for detection. Two hypotheses
30 AM, MET, RIF, AMX, ER 0.714 are supported by the presence of H. pylori DNA in river
31 AM, MET, RIF, AMX, CLR 0.714 water, well water, wastewater, and surface and shallow
Average 0.687 groundwater: either H. pylori is a waterborne organism
MAR index = No. of resistance / Total No. of tested antibiotics or water may become contaminated by the fecal-oral
route [25].
identification, particularly in clinical samples [33]. Addi- Helicobacter infection is a global zoonotic infection
tionally, Wongphutorn et al. [34] have recommended the with a high incidence, especially in Egypt [40]. In some
combination of several target genes for H. pylori detec- cases, H. pylori persists in the human host without caus-
tion, such as ureC and 16 S Rrna sequencing, to enhance ing illness. However, it has traditionally been linked to
diagnostic performance. various gastrointestinal illnesses due to induction of
Few authors have emphasized fish as a source of H. local and systemic inflammation [41]. Although human
pylori; Abdel-Moein et al. [14] could detect the organism infection with H. pylori has declined in many countries
in 1.9% of Tilapia fish. Pina-Pérez et al. [35] identified H. as a result of advances in treatment and improved living
pylori in various marine food products, including shell- standards, it still poses a significant risk in developing
fish. They verified the potential connection between the nations with overcrowded living conditions, inadequate
presence of H. pylori in seawater and the role of contam- sanitation, and unsafe drinking water. Therefore, inves-
inated seafood as means for H. pylori to enter the food tigating the prevalence rate in a large-scale population
is crucial, and various diagnostic methods for H. pylori
Mubarak et al. BMC Veterinary Research (2023) 19:250 Page 7 of 11

Fig. 2 Neighbor-joining phylogenetic tree of the obtained H. pylori isolates based on 16 S rRNA gene sequence

Fig. 3 Frequency of antimicrobial resistance of H. pylori isolates

are available. In our study, we examined 88 fish-han- healthy asymptomatic residents in the United Arab Emir-
dlers for the presence of H. pylori using the stool Ag ates as H. pylori positive. While higher detection rates
test, which yielded 40 positive samples (45.45%). This were observed in the Egyptian patients stools by Salem
method has been endorsed by many authors as rapid, et al. [7] and Shaaban et al. [44] at 73.7% and 74.8%,
reliable, and non-random [42]. A closely related result respectively, using the rapid antigen test, On the other
was obtained by Khoder et al. [43] who recorded 41% of hand, lower results were reported by Hassan et al. [45],
Mubarak et al. BMC Veterinary Research (2023) 19:250 Page 8 of 11

Almashhadany and Mayass [46], and Seid et al. [47] as 25, infection (17.65%); this result was proportionate with
30.4, and 18.45%, respectively. This disparity in H. pylori that obtained by Shah et al. [55], who recorded the low-
prevalence could be attributed to the sociodemographic est infection rate in the youngest age group 11–30 years
status of the investigated population, geographical varia- (19.8%). Nevertheless, different results were recorded
tions, and hygienic measures. On the other hand, the by previous studies, which were conducted by Khoder
results of the stool antigen test can be impacted by the et al. [43], who found that H. pylori infection increased
heterogeneity of the antigens, the serological procedures extremely with age, and Shaaban et al. [44], who recov-
used, and the amount of antigen present in the stool [48]. ered the highest infection rate in the over 50 years age
After culturing stool samples, the incidence rate of H. group (87.3%), followed by the 15–50 years age group
pylori has decreased to 39.77% (35/88). A compatible (76.8%). On the other hand, Mehesin et al. [42] demon-
result was obtained by Fabricio-Guaman et al. [49] who strated that there was no correlation between the preva-
obtained a lower incidence rate of H. pylori in patients by lence of H. pylori infection and age.
culture method (36%), than by antigenic screening test Along the same lines as other studies in terms of resi-
(50%). Contrarily, Abu-Gharbia et al. [50] recorded an dence, our findings indicated a higher prevalence of H.
incidence rate of H. pylori in patients attending Assiut pylori infection in rural residents (36.07%) than in urban
University Hospital and some laboratories at incidence residents (33.33%), with no significant difference. Salem
rates of 76.9% and 86.1% by stool antigen and culture et al. [7] stated that farmers are more likely to contract H.
methods, respectively. pylori infection (82.1%) than non-farmers (64.9%). Mehe-
PCR-based techniques are crucial for fast and accurate sin et al. [42] recorded an infection rate among rural and
identification of H. pylori infections among patients [51]. urban populations of 60.7 and 39.3%, respectively. Also,
So, in the present study, the ureC gene was amplified to Shaaban et al. [44] declared that the infection was more
confirm that the isolates yielded a 35.23% incidence rate. prevalent in those living in rural areas (88.5%) than in
Another point of this investigation includes the occur- those settling in urban areas (51.7%). This may be due to
rence of H. pylori in fish-handlers according to some risk the socio-economic status of the population, low edu-
factors such as sex, age, and residence. According to the cational level, poor hygienic conditions, and inadequate
current study results, a statistically significant correlation living resources. These factors facilitate the acquisition
between sex and the occurrence of H. pylori infection has of infection, as well as contact with animals, which are
been found. Based on molecular detection, males were considered a significant source of infection, as various
more infected (25, 48.08%) compared with females (6, studies have shown. This supports the theory that it is a
16.67%). Contrarily, a slightly higher infection rate was zoonotic disease.
detected in females (19.55%) than in males (16.44%) by The 16SrRNA gene displays a high level of functional
using an antigen screening test [46]. Through molecular and evolutionary homology within all bacteria, mak-
and serological studies, which were done by Castro et al. ing it a common choice for H. pylori detection [56]. In
[52], Mehesin et al. [42], and Shaaban et al. [44], there was this study, the sequencing of the 16SrRNA gene played
no significant higher infection rate in females than males, a major role in confirming our isolates as H. pylori, a
as 14.6, 11.2 and 55.5, 44.5 and 79.7, 65.5%, respectively. finding supported by Gong and El-Omar [57]. The phy-
Moreover, Rasheed et al. [53] and Wang et al. [22] were in logenetic analysis revealed that our isolates were closely
agreement with our result that males were more prone to related to reference strains found in the human gastro-
H. pylori infection than females. This could be explained intestinal tracts. This raises concerns about the poten-
by the greater exposure of males to environmental fac- tial role of the fecal-oral route in the transmission of this
tors as a result of working outside homes for longer peri- microorganism.
ods than females. According to the latest studies on the Increasing rates of H. Pylori resistance are associated
worldwide prevalence of H. pylori, there are no variances with a number of clinical issues; the gradual increase in
in H. pylori infection based on sex [54]. the number of antibiotic-resistant H. pylori strains and
This study included 88 fish-handlers at ages rang- their diverse resistance profiles makes treatment more
ing from 16 to more than 45 years old; the vast major- challenging and necessitates more pertinent research.
ity of the participants were 26–35 years old. The highest Furthermore, chromosome-encoded mutations are most
level of H. pylori infection was reported significantly in frequently attributed to H. pylori’s antibiotic resistance,
the 36–45 year old age group (46.67%), followed by the along with other factors such as membrane permeability
26–35 year old age group (39.53%), and then the age alterations, efflux systems, and biofilm formation [58].
group of ˃45 years. This can be explained by the nature of Our investigated H. pylori isolates were all resistant to at
this age; it is the age of working and so more contact with least three families of antibiotics. In addition, the high-
contaminated sources occurs. In our study, the youngest est resistance was seen against ampicillin, metronidazole,
age group (16–25 years) was less susceptible to H. pylori rifampicin, erythromycin, and amoxicillin, which are the
Mubarak et al. BMC Veterinary Research (2023) 19:250 Page 9 of 11

best choices for H. pylori treatment. This may be related 0.2, it means that the high-risk contamination source is
to the indiscriminate use of these antibiotics, which are the place where antibiotics are frequently used.
unfortunately frequently prescribed and in excess of rec-
ommended dosages. The antibiotics resistance mecha- Conclusions
nism can be explained by limiting drug uptake and The presence of H. pylori in fish is still unclear, despite
inactivation, modifying the drug target, or active drug its status as a major waterborne disease. To the best of
efflux [59]. In addition, there are other mechanisms such our knowledge, this work is precedent in assessing the
as mutational adaptations, acquisition of genetic material prevalence of H. pylori in fish, freshwater sources, and
or alteration of gene expression leading to resistance to fish-handlers. The use of the ureC gene as a genetic
almost all antibiotics [60]. marker plays a major role in identifying and confirming
All of the isolated strains were resistant to ampicillin the presence of H. pylori bacteria in our samples. In addi-
(100%); Ranjbar et al. [29] reported a somewhat lower tion, The creation of a phylogenetic tree revealed genetic
rate of resistance at 75%. Our H. pylori isolates exhibited relatedness among the isolates. This data supports the
a high resistance rate to metronidazole (95.24%). Abdoh hypothesis of a zoonotic route of H. pylori transmission.
et al. [61] and Metwally et al. [62] reported a resistance Therefore, there is a need to raise public awareness about
rate of 100%. Various rates of metronidazole resistance stopping the discharge of sewage into waterways to pre-
were obtained by several authors, including Ranjbar et al. vent water and fish pollution. Since our study identified a
[29] (62.5%), Brigitte et al. [63] (97.9%), Tang et al. [64] significant level of antibiotic resistance, increased efforts
(90.6%), Mégraud et al. [65] (58.6%), and Shrestha et al. from health officials are necessary to combat it. It is also
[66] (69%). The high resistance to this antibiotic may be crucial to implement health education programs and
attributed to its widespread use for many infectious dis- promote strict hygiene practices within households.
eases and its cheap price.
Abbreviations
Resistance of H. pylori isolates to rifampicin, erythro- H. pylori 
Helicobacter pylori
mycin, and amoxicillin was reported in various studies. ureC urease C gene
Ranjbar et al. [29] recorded the same resistance rates glmM phosphoglucosamine mutase
CLSI Clinical & Laboratory Standards Institute
against erythromycin and amoxicillin (62.5%). In the line MAR Multiple antibiotic resistance
with our high resistance level to rifambicin (88.10%),
Metwally et al. [62] detected a 90% resistance rate. In Acknowledgements
The Science, Technology, and Innovation Funding Authority (STDF) and the
contrast, H. pylori strains recovered by Mégraud et al. Egyptian Knowledge Bank (EKB) have partnered to provide open access
[67] were highly susceptible to amoxicillin, with only a funding. Authors’ contributions to the research.
1.2% resistance rate being detected.
Authors’ contributions
Among our strains, the lowest resistance rate was The authors A.G.M. and H.M.A.M. conducted several activities in their study,
recorded for levofloxacin (21.43%), which aligns with its including conceptualization, formal analysis, investigation, methodology
recommended use in the treatment of H. pylori infection. development, software implementation, validation, visualization, original
draft authoring, and reviewing and editing of the manuscript. The process of
Furthermore, several studies have reported zero resis- analysis and writing-reviewer/editing was conducted by H.H.A. which then
tance to levofloxacin [61, 63]. In contrast, Tang et al. [64], transitioned into the software development phase. The final version of the
Mégraud et al. [65], and Metwally et al. [62] reported manuscript was read and approved by all authors.

resistance rates of 28.2%, 17.6%, and 20% to levofloxacin, Funding

respectively. The Science, Technology, and Innovation Funding Authority (STDF), and
Among our strains, the macrolides, clarithromycin is the Egyptian Knowledge Bank (EKB) have partnered to provide open access
funding. Authors’ contributions to the research.
the most important agent for eradicating H. pylori infec- Open access funding provided by The Science, Technology & Innovation
tion. We detected a relatively low level of resistance to Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
clarithromycin in our study (26.20%). Similar results (EKB).

were detected by Shrestha et al. [66] (26%), while Brigitte Data Availability
et al. [63], Hofreuter et al. [68], and Mégraud et al. [67] Requests for materials should be addressed to A.G.M and H.M.A.M.
recorded lower rates of 13.75%, 10.9%, and 22.2%, respec- Accession numbers and [PERSISTENT WEB LINK]”.
OQ456179.
tively. In contrast, Abdoh et al. [61] identified high resis- https://www.ncbi.nlm.nih.gov/nucleotide/OQ456179.1?report=genbank&log$
tance levels at 47% and [64] (44.4%), respectively. The =nucltop&blast_rank=2&RID=HD49290W013.
resistance to Clarithromycin in H. pylori may be attrib- OQ456180.
https://www.ncbi.nlm.nih.gov/nucleotide/OQ456180.1?report=genbank&log$
uted to its misuse in the treatment of various respira- =nucltop&blast_rank=1&RID=HD4G6TMH013.
tory infections. Furthermore, a high MAR index of up to OR144424.
1 was observed in our isolates, indicating multiple drug https://www.ncbi.nlm.nih.gov/nucleotide/OR144424.1?report=genbank&log$
=nucltop&blast_rank=1&RID=HD4X692S01N.
resistance. These findings were corroborated by Rotchell OR144425.
et al. [69], who reported that when the MAR is more than
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