CHARACTERISTICS OF HELICOBACTER PYLORI INFECTION: RESISTANCE TO ANTIBIOTICS AND ASSOCIATION WITH HOST TLR1, PRKAA1, PSCA, MUC1 AND PLCE1 GENE POLYMORPHISMS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Gintarė Dargienė

CHARACTERISTICS OF HELICOBACTER

PYLORI INFECTION: RESISTANCE TO

ANTIBIOTICS AND ASSOCIATION WITH

HOST TLR1, PRKAA1, PSCA, MUC1 AND

PLCE1 GENE POLYMORPHISMS

Doctoral Dissertation Medical and Health Sciences,

Medicine (M 001)

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The dissertation was prepared in the Medical Academy of Lithuanian University of Health Sciences during the period of 2014–2020.

Scientific Supervisor

Prof. Dr. Juozas Kupčinskas (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001).

Dissertation is defended at the Medical Research Council of the Medical Academy of Lithuanian University of Health Sciences:

Chairperson

Prof. Habil. Dr. Virgilijus Ulozas (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001).

Members:

Prof. Dr. Astra Vitkauskienė (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001);

Assoc. Prof. Dr. Rasa Liutkevičienė (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001);

Prof. Dr. Ligita Jančorienė (Vilnius University, Medical and Health Sciences, Medicine – M 001);

Prof. Dr. Juris Pokrotnieks (Riga Stradins University (Latvia), Medical and Health Sciences, Medicine – M 001).

Dissertation will be defended at the open session of the Medical Research Council of Lithuanian University of Health Sciences on the 30th_{of June 2020}

at 2 p. m. in the Great Auditorium at the Hospital of Lithuanian University of Health Sciences Kauno klinikos.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Gintarė Dargienė

HELICOBACTER PYLORI INFEKCIJOS

YPATUMAI: ATSPARUMAS

ANTIBIOTIKAMS IR SĄSAJA SU

ŽMOGAUS TLR1, PRKAA1, PSCA, MUC1

IR PLCE1 GENŲ POLIMORFIZMAIS

Daktaro disertacija

Medicinos ir sveikatos mokslai, medicina (M 001)

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Disertacija rengta Lietuvos sveikatos mokslų universitete Medicinos akade-mijoje 2014–2020 metais.

Mokslinis vadovas

prof. dr. Juozas Kupčinskas (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Medicinos akademijoje medicinos mokslo krypties taryboje:

Pirmininkas

prof. habil. dr. Virgilijus Ulozas (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001).

Nariai:

prof. dr. Astra Vitkauskienė (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001);

doc. dr. Rasa Liutkevičienė (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001);

prof. dr. Ligita Jančorienė (Vilniaus universtitetas, medicinos ir sveikatos mokslai, medicina – M 001);

prof. dr. Juris Pokrotnieks (Rygos Stradiņio universitetas (Latvija), medicinos ir sveikatos mokslai, medicina – M 001).

Disertacija ginama viešame Lietuvos sveikatos mokslų universiteto Medici-nos mokslų krypties tarybos posėdyje 2020 m. birželio 30d. 14 val. Lietuvos sveikatos mokslų universiteto ligoninės Kauno klinikų Didžiojoje audito-rijoje.

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ABREVIATIONS

AG – atrophic gastritis

AMPK – adenosine monophosphate-activated protein kinase aOR – adjusted odds ratio

Arg – arginine

ATC – Anatomical Therapeutic Chemical ATP – adenosine triphosphate

CI – confidence interval DAG – diacylglycerol DDD – Defined Daily Dose DNA – deoxyribonucleic acid

dNTP – deoxyribonucleoside triphosphates EC – esophageal cancer

EDTA – Ethylene Diamine tetra acetate (ethylenediaminetetraacetic acid) ESCC – esophageal squamous cell carcinoma

ESPGHAN – European Society for Pediatric Gastroenterology Hepatology and Nutrition

EUCAST – European Committee of Antibiotic Susceptibility Testing

GC – gastric cancer

GCA – gastric cardia adenocarcinoma GWAS – genome-wide association studies

His – histidine

HRAG – high risk atrophic gastritis HWE – Hardy-Weinberg equilibrium IC – internal positive control

IL – interleukin

IP3 – inositol 1,4,5-triphosphate LKB1 – liver kinase B1

LPS – lipopolysaccharides

M – Molar mass

MALT – mucosa-associated lymphoid tissue MAMPs – microbe-associated molecular patterns

McF – McFarland

mTORC1 – rapamycin complex 1

MUC1 – Mucin 1

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NASPGHAN – North American Society for Pediatric Gastroenterology Hepatology and Nutrition

NF-κB – nuclear factor kappa-light-chain-enhancer of activated B cells

nm – nanometers

OR – odds ratio

PBPs – penicillin binding proteins PCR – polymerase chain reaction PLCE1 – phospholipase C epsilon 1 PPI – proton pump inhibitors

PRKAA1 – protein kinase AMP-activated alpha 1 catalytic subunit PRRs – pattern recognition receptors

PSCA – prostate stem cell antigen QC – quality control

RCF – relative centrifugal force rpm – revolutions per minute

RT-PCR – real time polymerase chain reaction SD – standard deviation

SNP – single nucleotide polymorphism

spp. – species

TLR1 – toll-like receptor 1 TNFα – tumor necrosis factor – α UV – ultraviolet light

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INTRODUCTION

Helicobacter pylori (H. pylori) infection is the major known infectious

agent in gastric diseases [1]. More than 50% of the global population is estimated to be infected [2]. H. pylori infection in the stomach leads to chronic inflammation, atrophic gastritis (AG), peptic ulcer disease and gastric adeno-carcinoma or mucosa-associated lymphoid tissue lymphoma (MALT lymphoma) [3, 4]. Furthermore, in a considerable number of individuals

H. pylori infection is associated with dyspeptic symptoms [1]. Despite

de-creasing prevalence of H. pylori infection in developed countries, it still has significant clinical implications and eradication can prevent long-term compli-cations [3]. Since discovery of H. pylori in 1982 a huge amount of publica-tions was published (44,119 papers in PubMed till Feb 1st_{, 2020), however}

not all scientific and practical problems concerning the infection was solved. In our work we concentrated on research of two characteristics of

H. pylori: 1) regional resistance of bacteria to antibiotics; and 2) association

with host genetic predisposition to infection.

Regular and periodical investigation of the resistance of H. pylori to the most commonly used antimicrobial agents in the different countries is necessary according to the Maastricht V/Florence Consensus Report [5]. In order to identify the most effective empirical eradication strategies in Lithua-nia, H. pylori susceptibility testing has been periodically carried out in the acknowledged H. pylori investigation center of Gastroenterology Clinic (Hospital of Lithuanian University of Health Sciences Kauno klinikos) since 1998.

Increasing H. pylori resistance to previously efficacious antibiotic regi-mens is essentially driven by the direct exposure of patients to antimicrobial agents [3,6]. The knowledge of antibiotic consumption in a given region may provide a simple tool to predict the susceptibility of H. pylori to quinolones and macrolides, and to adapt the treatment strategies in settings where diagnostic laboratory facilities are not available [6]. Therefore, one of our objections was to investigate the trends of antibiotic consumption in Lithuania during the 12-year period (2003–2015).

To date it is unclear why certain part of population is never infected with

H. pylori, even in the presence of high exposure rates. It is plausible that

certain host genetic predisposition is associated with susceptibility to H. pylori infection. This theory is supported by Genome – wide association study (GWAS) in European descent population that has identified the association of rs4833095 in TLR1 with H. pylori seroprevalence [7].

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What is more, among the infected individuals, the severity of H. pylori related diseases varies greatly, with the outcome governed by interactions between host genetic [8], epigenetic [9–12], apoptotic pathways [13] or environmental factors [14]. H. pylori induced chronic atrophic gastritis is the most consistent risk factor for development of gastric cancer (GC) [1]. However only a small proportion of individuals, who are infected with

H. pylori and are exposed to similar risk factors, develop GC [15]. Therefore,

host genetic factors might be an important component in the pathogenesis of

H. pylori related diseases, including GC.

Aim of the study

To evaluate primary antibiotic resistance rates of H. pylori strains in Lithuania and to investigate association between the host single nucleotide polymorphisms of PSCA, MUC1, PLCE1, TLR1, PRKAA1 genes and H.

pylori infection.

The objectives of the study

1. To evaluate primary antibiotic resistance of H. pylori strains in children and adults in Lithuania during the period 2013–2015. 2. To investigate the consumption of antibiotics used for H. pylori

eradication regimens in Lithuania during the period 2003–2015. 3. To investigate associations between the host single nucleotide

polymorphisms of PSCA, MUC1, PLCE1, TLR1, PRKAA1 genes and H. pylori infection.

4. To investigate associations between the host single nucleotide poly-morphisms of PSCA, MUC1, PLCE1, TLR1, PRKAA1 genes and diseases related to H. pylori infection – atrophic gastritis and gastric cancer.

Novelty of the study

Eradication of H. pylori in infected patients is the most important goal in the management of H. pylori-associated diseases [3]. The prevalence of bacte-rial antibiotic resistance is regionally variable and appears to be markedly increasing with time in many countries [16]. According to the Maastricht V/Florence Consensus Report, the treatment regimen should be chosen according to the resistance rates of clarithromycin and metronidazole. PPI-clarithromycin-containing triple therapy without prior susceptibility testing should be abandoned when the clarithromycin resistance rate in the

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region is more than 15% [3]. In this study we present the data about the primary antibiotic resistance of H. pylori strains in adults and children within the period 2013–2015 and the trends of antibiotic consumption during the period 2003–2015 in Lithuania. This data is essential while making therapeu-tic choice of primary empirical H. pylori eradication therapy. Thereby in the study we obtain the new data about H. pylori regional resistance during the year 2013–2015.

Genome wide association studies (GWASs) have permitted the identify-cation of novel, high prevalence, low-penetrance genetic variations in the human genome associated with common diseases, including cancer [17]. Nevertheless, the results of GWASs need replication in independent cohorts of patients in different populations for validation of these findings, due to the large number of traits analyzed.

Since single nucleotide polymorphisms (SNPs) varies greatly in percent-age among populations of different ethnicities the role of each SNP in risk prediction for various diseases might be divergent in different populations.

All of the SNPs investigated in our study were widely analyzed in Asian population, but studies in European descent population were scarce. To our best knowledge, this is the first replication study in European population of the genetic variant TLR1 (rs4833095) identified in H. pylori susceptibility GWAS. It must be pointed out that TLR1 (rs4833095) and PRKAA1 (rs13361707) SNPs also have not been investigated previously in patients with H. pylori induced gastritis.

One of the major findings of this study was the significant association of

PSCA and MUC1 SNPs not only with GC, but also with its precursor states,

elucidating potential genetic predisposition that might be involved in early stages of GC development. What is more, we found that TLR1 SNP (rs4833095) was significantly associated with GC in a European descent population; however, this SNP was not linked with the susceptibility to

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1. REVIEW OF LITERATURE

1.1. H. pylori: epidemiology and related diseases

In 1982 Marshall and Warren identified and subsequently cultured the gastric bacterium, Campylobacter pyloridis, later reclassified as Helicobacter

pylori [18]. H. pylori is a spiral, Gram-negative, microaerophilic pathogen

with a strict tropism for the gastric mucosa [19, 20].

It is the most common chronic bacterial infection in humans [21].

H. pylori prevalence is ranging from 50.8% in developing countries to 34.7%

in developed countries. Overall, the trend of declining prevalence of H. pylori infection is continuing, with the major evidence available from studies in Europe [21]. A large multicenter study in the Czech Republic demonstrated the decrease in H. pylori prevalence during the ten year period: from 41.7% (in 2001) to 23.5% (in 2011) [22]. According to Tacikowski and colleagues the current prevalence of H. pylori infection among patients with dyspepsia treated in Poland is 35.8%. The prevalence had decreased almost two times compared to the previous Polish studies [23]. H. pylori seropositivity was reported in 28.9% of German blood donors from Magdeburg, meaning that the prevalence of H. pylori had decreased compared to the previous study in 2010. (H. pylori seroprevalence of 44.4 % in patients at the emergency ward of the University Hospital of Magdeburg) [24]. However Baltic countries are still denoted as the area of high H. pylori prevalence [25]. A number of systematic reviews and meta-analyses that have been published during the past year, indicates the lowest prevalence rates of infection in Oceania (24.4%), the highest in Africa (79.1%) [21]. Differences in prevalence exist within and between countries, with higher prevalence seen among people with lower socio-economic status. Most transmission of infection occurs early in life [2]. The reappearance of H. pylori in a previously treated patient can occur through two distinct mechanisms: recrudescence and re-infection. Recrudescence involves the reappearance of the original H. pylori strain after its temporary suppression, indicating that it was not effectively eradicated, while reinfection occurs when, after successful eradication, a patient is infec-ted with a new strain of H. pylori [19]. Reinfection with H. pylori following successful bacterial cure is unusual [26–28]. The global annual recurrence (either by recrudescence or reinfection) rate of H. pylori is 4.3%. It is directly related to the human development index and prevalence of infection [21].

Once acquired, infection persists and may be the cause of upper gastro-intestinal as well as extra gastric diseases [29]. H. pylori is able to colonize and adhere to the gastric epithelium through several mechanisms, including

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the breakdown of urea with production of the cell-toxic ammonia. The re-sulting raise in pH neutralizes acidity of the stomach, thereby allowing the bacterium to safely cross the mucus layer to the epithelial surface [20].

H. pylori remains the main etiopathogenetic factor in complicated and

uncomplicated peptic ulcer disease [29]. What is more, the adhesion to host’s gastric mucosa of H. pylori can cause transition from normal mucosa to chronic superficial gastritis, which then increases the risk of atrophic gastritis and intestinal metaplasia, and eventually leads to the development of gastric cancer in some of the infected individuals over many years [30]. H. pylori infection is a well-established risk factor in gastric cancerogenesis and is classified as a Class I human carcinogen by the World Health Organization (WHO) based on epidemiological evidence [31]. Eradication of H. pylori infection has been proven to reduce the incidence of gastric cancer [32]. Although the involvement of H. pylori in functional dyspepsia is contro-versial, several data support the importance of H. pylori – induced gastritis in the pathogenesis of dyspeptic symptoms in adults. Recent interventional studies have reported that H. pylori eradication improves dyspepsia mainly in areas with a high prevalence of this bacterium [29]. On the other hand, studies in children do not support a role for H. pylori infection in functional disorders such as recurrent abdominal pain [33]. There is evidence that H. pylori in-fection is a risk factor for gastric MALT lymphoma [34]. What is more, many studies have demonstrated a potential role of H. pylori in the pathogenic mechanisms of different extragastric diseases [35]. It might be associated with idiopathic iron deficiency anemia, vitamin B12 deficiency, and idiopathic

thrombocytopenic purpura [3, 4, 34]. Although, in order to confirm causal role further studies are still needed, there is increasing evidence of a potential role of H. pylori in many other diseases such as cardiovascular, neurologic, metabolic and colorectal disorders [35].

1.2. H. pylori resistance to antibiotics

Development of H. pylori therapy differs from other infectious diseases. Since the advent of antibiotics, infectious diseases therapy has been suscep-tibility based, whereas most H. pylori treatment guidelines for adults recom-mend susceptibility testing only after two empiric therapy failures [36]. On the other hand, for pediatric patients it is recommended to obtain antimicro-bial susceptibility for the infecting H. pylori strains and to tailor eradication therapy accordingly. Since antimicrobial susceptibility testing is not available in all centers, the national/regional effectiveness of H. pylori eradication regimens in children and adolescents should be evaluated, if possible [33]. If

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susceptibility testing is carried out, it is important to take biopsies for

H. pylori culture from different parts of the stomach: antrum and corpus.

According to the study by Selgard et al., antibiotic resistance varies between the antrum and corpus of the stomach for both: H. pylori therapy-naive and pre-treated patients. Therefore, resistant H. pylori strains may be missed if just one biopsy from one anatomic site of the stomach is taken for H. pylori susceptibility testing [37].

In the last 20 years, the recommended regimens for H. pylori eradication have included the combination of a proton-pump inhibitor and two or more antibiotics [20]. However the efficacy of the H. pylori eradication treatment has decreased dramatically because of antibiotic resistance [1, 3, 32]. Identifi-cation of reliable empirical H. pylori eradiIdentifi-cation therapy has proved difficult, partly because brief exposure of H. pylori to commonly used antibiotics such as macrolides, nitroimidazoles or quinolones often results in resistance (bystander effect) [36]. What is more, data concerning the time lag between antibiotic use and shift in antibiotic resistance are lacking. H. pylori consti-tutes a paradigm of long-lasting infection and it should be stressed that the time of antibiotic exposure of this organism to antibiotics may be much longer than for most other pathogens [6]. The four main mechanisms of antibiotic resistance are: 1) lack of antibiotic penetration (may concern all antibiotics), 2) modification of the target (macrolides, quinolones and β-lactams), 3) enzy-matic inactivation of the antibiotic (β-lactams) and 4) elimination after pene-tration (tetracycline) [20]. The molecular basis of drug resistance in H. pylori has been found to result from the drug efflux mechanism or can be attributed to the presence of mutations. The main H. pylori resistance mechanism results from the acquisition of point mutations. With regard to clarithromycin, the mutation mechanism is mainly in the 23S rRNA region, while the amoxicillin resistance acquisition is associated with mutation in the penicillin binding proteins (PBPs), and metronidazole and levofloxacin are associated with a mutation in the rdxA and gyrA genes respectively [19].

In addition to the mechanisms of resistance developed by H. pylori to the main antimicrobials used in the treatment of infection of the bacteria, bacterial virulence factors have been associated with resistance: less virulent (cagA-negative and vacA S2-containing) strains of H. pylori are associated with primary clarithromycin resistance [38]. According to the study by L-J van Doorn et al., cure rates were higher for patients with cagA-positive/vacA s1

H. pylori strains [39]. H. pylori cagA-positive strains are more susceptible to

antimicrobial activity than negative cagA strains, because antibiotics are most active on the bacteria that grows quickly, and H. pylori strains carrying the cagA gene are at a higher density and have an intense inflammatory response

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in the gastric epithelial cells and, thus, can proliferate more rapidly than cagA negative strains [19].

The European multicenter study from 32 centers in 18 European count-ries showed a steady increase in clarithromycin resistance and a rapid emer-gence of levofloxacin resistance in H. pylori. Among adults, resistance rates of H. pylori were reported to be 17.5% for clarithromycin, 14.1% for levoflo-xacin and 34.9% for metronidazole, while in children the rate of clarithro-mycin resistance was even higher often exceeding 30% [6]. Despite the increasing resistance to clarithromycin all over the Europe, the Northern countries such as Finland (4%), Norway (9%), Ireland (9.3%), United Kingdom (9%) stand out with lower resistance rates in adult population. [6, 40–42] This is in contrast to much higher prevalence of clarithromycin resi-stance in Southern European countries: Italy (35.2%), Spain (17.9%), Portu-gal (21.4%) or France (22.2%) [43–46]. Therefore, it could be stated, that traditionally, in Northern Europe primary resistance rates of H. pylori to clari-thromycin has always been lower compared to Western/Central and Southern Europe [6]. Reported H. pylori quinolone resistance rates in different count-ries vary a lot: Poland 5.9%, Austria 10.2%, Germany 11.9%, Finland 12%, Spain 13.9%, France 15.4%, Italy 22.1%, Portugal 26.2% [40, 43–49]. The published H. pylori antibiotic resistance studies in adult population greatly outnumber those in pediatric population [32]. Nevertheless, according to available data, the prevalence of clarithromycin and metronidazole resistance in pediatric population was 11.9% and 10.1% in Croatia, 23.2% and 28.7% in Germany, 26% and 33% in Italy, 34.7% and 16.7% in Spain respectively. Furthermore, there were either no H. pylori resistance to amoxicillin detected (Italy and Spain), or it was very low (Croatia – 0.6% and Germany 0.8%) [50–53]. Studies from other areas of the world have also clearly shown an increasing resistance of H. pylori, with clarithromycin resistance as high as 30% in Japan, 50% in China and 40% in Turkey [16]. According to systemic review and meta-analysis on prevalence of antibiotic resistance in H. pylori in World Health Organization (WHO) regions by Savoldi et al. resistance to clarithromycin, metronidazole and levofloxacin was found to cross the thre-shold of 15% in the majority of WHO regions, meanwhile primary resistance to amoxicillin and tetracycline was ≤10% everywhere except in Eastern Mediterranean region, where amoxicillin resistance reached 14% [32].

The previous study in Lithuania by Kupcinskas Limas et al. showed that during a ten year period (year 1998–2008), there were no significant changes in H. pylori susceptibility in adult population to the most widely used anti-biotics for H. pylori eradication: resistance rates to metronidazole were high (35.6%), no resistance to amoxicillin was detected, and resistance rates to

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clarithromycin (3.3%), ciprofloxacin (5.6%) and tetracycline (2.5%) – remai-ned low [54]. Meanwhile, primary H. pylori resistance rates in pediatric population for the first time was investigated in Lithuania during the year 2007–2008 by Kupcinskas L. and colleagues. According to the study the primary H. pylori resistance rates for children were: for metronidazole – 20.7%, for clarithromycin – 16.8%, and no cases of amoxicillin, tetracycline, ciprofloxacin were detected [55].

1.3. Association of H. pylori resistance with outpatient antibiotic consumption

The alarming global levels of H. pylori resistance in treatment-naive patients can be correlated with the increasing and uncontrolled consumption of antibiotics that are commonly used in H. pylori empirical therapy and also used to treat other common infections in the general population [32]. Specific

SNP (single nucleotide polymorphisms) are the main molecular basis of drug

resistance in H. pylori infections and the pressure that determines the selection of resistant strains is related to frequent and/ or inappropriate use of antibiotics [19]. According to the study of outpatient antibiotic use in 26 countries in Europe prescription of antibiotics in primary care in Europe varied greatly; the highest rate was in France (32.2/DDD per 1000 inhabi-tants/day) and the lowest was in the Netherlands (10/DDD per 1000 inha-bitants/day). What is more, there was a significant differences across Europe, with antibiotic use being low in northern, moderate in eastern, and high in southern regions [56]. Extreme geographical variations, which was observed not only for overall prescription but also for the use of various specific anti-biotic subgroups including quinolones, macrolides, penicillins, and cepha-losporins suggest that they are to a large extent inappropriately used [57, 58]. The European multicenter study by Megraud Francis et al. in 18 European countries revealed that the prevalence of clarithromycin resistance in

H. pylori was positively associated with national levels of macrolide

con-sumption. What is more, a strong association was found between quinolone use (mainly ciprofloxacin, except in Italy where levofloxacin was mostly used) and the proportion of resistance of H. pylori to levofloxacin [6]. Kenyon analyzed available data for macrolide consumption and resistance in 31 and 52 countries, respectively and found that the prevalence of clarithromycin resistance was positively correlated with macrolide consumption in the 29 countries (with data available for both variables) [59].

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1.4. TLR1 gene in H. pylori seroprevalence and H. pylori related diseases

Epithelial cells of gastric mucosa are amongst the first cellular barriers for H. pylori in the gastrointestinal tract as they recognize H. pylori-derived microbe-associated molecular patterns (MAMPs) through ligation of pattern recognition receptors (PRRs), especially the TLRs. The PRRs recognize bacterial lipopolysaccharides (LPS) to effect the secretion of pro-inflamma-tory molecules [60].

GWAS revealed significant association of SNPs in TLR1 gene with susceptibility of H. pylori in European descent population [7]. This study was replicated in Thai population: the TLR1 (rs4833095), C allele was associated with significantly increased risk for H. pylori infection [61]. Interestingly, in a Chinese population with high risk of gastric cancer (GC) TLR1 (rs4833095) and TLR10 (rs10004195) were associated with decreased risk of H. pylori infection and precancerous gastric lesions [62]. Other study, performed in Malaysian population, showed that SNPs of TLR1 (rs4833095) and TLR10 (rs10004195) were directly associated with H. pylori infection, and conferred susceptibility to development of gastrointestinal disease, especially GC in

H. pylori infection [60]. This could be explained by hypothesis that H. pylori

is initially sensed by TLR signaling and polymorphisms in TLR components modulate the magnitude of resultant immune responses. The different outco-mes of inflammatory response are likely mediated by the downstream expres-sion of the key inflammatory cytokines, especially interleukin – 1β (IL-1β), IL-1α, IL-6, IL-8, IL-10, tumor necrosis factor – α (TNFα), and interferons, which have been shown to be varied when different genetic polymorphisms of TLR genes are considered [60, 61].

1.5. PRKAA1 gene in gastric cancer

The rs13361707 SNP is located in the first intron of PRKAA1 on locus 5p13.1. PRKAA1 is a gene that encodes AMPK (adenosine monophosphate-activated protein kinase), but the exact function of this SNP polymorphism has not been determined [63]. AMPK is a key regulator of the balance bet-ween cell growth and bioenergetic homeostasis. In general, AMPK promotes processes that generate or preserve cellular ATP, including glycolysis, oxi-dative phosphorylation, β-oxidation of fatty acids and autophagy, and inhibits processes that consume ATP, such as protein translation, ribosome assembly and lipid synthesis [64]. AMPK activation by LKB1 (liver kinase B1), during energy stress, prolongs cell survival by maintaining NADPH and ATP levels thus cancer cell is protected. LKB1 – deficient or AMPK – deficient cells are

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resistant to oncogenic transformation and tumorigenesis, possibly because of the function of AMPK in metabolic adaptation [65]. On the other hand, AMPK plays a role in tumor suppression because of its ability to inhibit mTORC1 (rapamycin complex 1) and almost all biosynthetic pathways required for cell growth, and to cause cycle arrest [66]. A growing number of studies thus suggest a duality of functions for AMPK that are either pro- or anti-cancer, depending on context [64]. Some studies, investigating the function of AMPK, have focused on its critical role in the development of cancers and on its potential use as a therapeutic target for some malignant tumors [67]. It is possible that AMPK plays an important role in gastric cancerogenesis and, therefore, polymorphic alleles of the encoding gene could modify individual susceptibility to GC [68].

GWAS identified that the SNP in PRKAA1 (rs13361707) gene was associated with non-cardia GC in Chinese descent patients [69]. What is more, other studies also showed significant gene-based association between

PRKAA1 and GC [68, 70–73]. Moreover, one study found that PRKAA1

polymorphisms may have an influence to H. pylori infection development and synergetic effect on risk of development of GC [74]. Furthermore, association between PRKAA1 and GC was identified in European population in GWAS in Iceland [73]. In contrast, the results of meta-analysis by Jianfeng and col-leagues showed that PRKAA1 (rs13361707) was not significantly associated with GC risk in Asian population, despite few positive results in the subgroups [75]. Since the results of different studies were ambiguous, Jiang and colleagues performed meta-analysis (in 2018) of fifteen independent case – control studies, with a reasonable number of GC patients and control subjects. The overall analysis indicated that PRKAA1 rs13361707 polymor-phism significantly increased susceptibility for GC in all genetic models [67].

1.6. PSCA gene in gastric cancer and pre-malignant gastric conditions

PSCA (prostate stem cell antigen) is a 123-amino acid cell membrane

glycoprotein that belongs to the LY-6/thy-1 family of cell surface antigens [76]. PSCA gene is located on chromosome 8q24.2 and it encodes a glycosyl-phosphatidylinositol-anchored protein [77]. The precise function of PSCA in

vivo is not known [78]. It is expressed in the epithelium of several organs,

such as prostate, bladder, gallbladder and stomach. PSCA is considered to be involved in the cell proliferation inhibition and (or) cell death induction [79].

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PSCA was first identified as a prostate – specific antigen overexpressed

in prostate cancer [77]. Later, other studies indicated that PSCA was up-regu-lated in other human solid tumors such as pancreas, bladder, ovary, and renal cell [78, 80]. On the other hand, the expression of PSCA is absent or reduced in esophageal and gastric cancers [78]. Interestingly in the normal gastric epithelium, the main expression site is the isthmus and neck regions, which contains differentiating cells. In particular the expression of PSCA is down-regulated in gastric tissue with intestinal metaplasia [81].

Previous GWASs revealed the association of SNPs in PSCA with two H.

pylori related diseases: duodenal ulcer and GC. In Japanese GWAS the SNP

(rs2294008) in PSCA was associated with increased risk of duodenal ulcer [82]. Another GWAS in Japan identified two SNPs (rs2976392 and rs2294008) in the PSCA gene as the susceptibility loci of GC, especially in diffuse type [81]. Since then various replication studies were performed in different ethnic groups [74, 78, 79, 83–91]. However, the results were inconsistent with speci-fic region and population, GC sites and histological types. Zhao and collea-gues performed a case control study in different populations in China. Inte-restingly, only H. pylori negative non-cardia GC patients were included in the study. The results showed that rs2294008 may differently contribute to GC among different nationalities in one area and its role is independent from

H. pylori infection [84]. The study by Toyoshima and colleagues revealed

that rs2294008 was associated with the progression to chronic gastritis but not with H. pylori infection per se nor the progress from active gastritis to GC. PSCA expression was decreased in severe gastritis compared with mild gastritis only among T allele carriers [92]. Several meta-analysis regarding

PSCA SNPs (rs2294008 and rs2976392) and GC susceptibility were

conducted in order to resolve the controversy [93–98]. A large – scale meta-analysis involving 16 studies with overall 18 820 GC cases and 35 756 cont-rols was performed by Xixi Gu and colleagues in 2013. According to the meta-analysis, polymorphisms rs2294008 and rs2976392 of PSCA was a risk – conferring factor associated with increased GC susceptibility. However, in stratified analysis by ethnicity, significantly increased risks were found for rs2294008 and rs2976392 polymorphism among East Asians in all genetic models, while no significant associations were observed for the rs2294008 polymorphism in Caucasians [96]. The most recent meta-analysis by Yong Gu and colleagues included 32 studies consisting 30 028 cases and 38 765 controls for the rs2294008 polymorphism, and 14 studies with 8190 cases and 7176 controls for the rs2976392 polymorphism. The authors found that PSCA rs2294008 polymorphism was associated with increased overall cancer risk. The stratification analysis by cancer types found that carriers of PSCA rs2294008 T had a significantly increased risk of GC and increased risk of

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bladder cancer. What is more, this polymorphism increased cancer risk among Asians, Caucasians as well as Africans. Similarly PSCA rs2976392 polymorphism was associated with increased overall cancer risk and the significant association was observed for GC [97]. Therefore given the important roles of PSCA in the regulation of cell adhesion, proliferation and survival, it is biologically plausible that genetic variations of PSCA polymor-phisms may modulate the risk of GC [95,97].

1.7. MUC1 gene in gastric cancer and pre-malignant gastric conditions

MUC1 (Mucin 1, encoded by MUC1 gene, located on chromosome 1q22) is a membrane-bound extracellular protein anchored to the apical surface of gastrointestinal epithelia by transmembrane domain [99]. It is a multifunc-tional protein involved in mucosal lubrication, protection from pathogens, signal transduction and cell-cell interaction [100]. Functional studies showed that rs4072037, in the MUC1 gene, regulates alternative splicing of the se-cond exon, and modifies the transcriptional activity of the promoter. MUC1 participates intracellularly in the activation of the NF-κB, leading to up-regula-tion of cytokines IL-6 and TNFα [101, 102]. What is more, rs4072037 polymorphism was observed to disrupt the physiological function of MUC1, which is important for protection of the gastric mucosa This implies that SNP rs4072037 is a potential genetic factor leading to increased susceptibility to GC by altering MUC1 [103]. MUC1 is over-expressed in breast, ovarian, lung, pancreatic and prostate cancers [100].

Susceptibility to H. pylori gastritis and gastric cancer appears to be associated with MUC1 allele length. MUC1 binds to H. pylori, Muc1 knock-out mice are susceptible to H. pylori gastritis and there is an altered pattern of expression of MUC1 in H. pylori gastritis [102].

Abnet and colleagues conducted a GWAS of GC and esophageal squamous cell carcinoma (ESCC) in ethnic Chinese subjects in the year 2010. The researchers observed a near-significant association between rs4072037 G allele and reduced GC risk in the first phase of their GWAS. But no significant result was obtained in the second phase [104]. A large sample size case – control study in Japan identified that rs4072037 was only associated with diffuse type GC but not intestinal type GC [100]. Nevertheless subse-quent case-control studies showed significant association of MUC1 (rs4072037) and decreased risk of GC [17, 90, 101, 103, 105]. A case – control study by Li and colleagues reported that the most relevant GC risk was found in subjects with H. pylori seropositivity and AA genotype of

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rs4072037 compared with those with H. pylori seronegativity and AG or GG genotypes, showing that this SNP may interact with H. pylori infection to increase the risk of GC [31]. Following meta-analyses showed that the G allele of MUC1 rs4072037 was significantly associated with decreased risk of GC [99, 106–110]. However, in stratified analysis for the ethnicities the results were inconsistent. Some of the meta-analyses detected the association of SNP rs4072037 with GC susceptibility in Asian rather than Caucasian population [106, 107, 110]. Others, indicated that rs4072037 decreased the risk of GC more among Caucasians than Asians [108, 109]. What is more subgroup analyses also revealed that MUC1 (rs4072037) was associated with both GC subtypes: diffuse and intestinal [99, 107, 109].

1.8. PLCE1 gene in gastric cancer and pre-malignant gastric conditions

PLCE1 (phospholipase C epsilon 1), located at 10q23 encodes a

pho-spholipase enzyme that catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate to generate two second messengers (inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG)). These second messengers subsequently regulate various processes affecting cell growth, differentiation, and gene expres-sion [111]. Rs2274223 A>G SNP causes a missense variation (His>Arg) in PLCE1 protein. Recent investigations demonstrated that PLCE1 might have a role in gastric cancerogenesis as it is overexpressed in precancerous chronic atrophic gastritis tissue and GC, compared with normal stomach tissue, and its inhibition has therapeutic potential in a xenograft activity in some animal models [112].

PLCE1 (rs2274223) was identified as a genetic marker for both gastric

cardia cancer and esophageal squamous cell carcinoma susceptibility by two GWASs in a Chinese population [104,113]. Furthermore PLCE1 (rs2274223) was associated with a significantly increased risk of head and neck cancer [114]. Meta-analysis by Zhang and colleagues indicated that PLCE1 (rs2274223) was significantly associated with increased risk of digestive tract cancer, especially among Asian population [115]. A number of individual association studies, replicating the findings of the above mentioned GWAS, reported inconclusive results [17, 90, 101, 116, 117]. Therefore, in order to obtain a comprehensive conclusion, several meta-analyses were performed. Accor-ding to the meta-analysis by Xue and colleagues, PLCE1 rs2274223 polymor-phism was associated with an increased risk of overall cancer, especially ESCC other than GC, especially among subgroups of Asian [118]. Umar and colleagues also reported that in their meta-analysis PLCE1 rs2274223

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polymorphism was significantly associated with increased cancer risk, but stratified analysis showed elevated risk of only GC and EC. Sub-group analysis based on ethnicity suggested PLCE1 polymorphism conferred signi-ficant risk among Asians but not Caucasians [119]. The results of other two meta-analysis indicated that PLCE1 rs2274223 G allele significantly contri-buted to the risk of ESCC and gastric cancer adenocarcinoma, especially in Chinese population [120, 121]. According to the comprehensive study on the published evidence about the association between genetic variants and risk of GC by Mocellin and colleagues, when all available datasets, Asian and Caucasian ancestry, were included in the meta-analysis, the G allele was significantly associated with increased risk of GC with the low level of evidence due to a high degree of between-study heterogeneity. Meanwhile the meta-analysis of only Asian ancestry subjects showed the G allele of rs2274223 to be associated with an increased risk of cardia GC, with a strong level of evidence. What is more, according to the meta-analysis rs2274223 was not associated with non-cardia GC with a high level of evidence, suggesting that this SNP is specifically linked to the carcinomas originating in the cardia region of the stomach [98].

Interestingly, GC survival studies, demonstrated that PLCE1 rs2274223 G allele may be associated with improved GC patient survival. A study by Dewei and colleagues, revealed, that patients carrying PLCE1 rs2274223 AA genotype survived for a significantly shorter time than those carrying AG and GG genotypes [122]. What is more Gu and colleagues also found that rs2274223 in PLCE1 was significantly associated with increased GC survi-val, especially in the early-onset subgroup [123]. These studies propose that rs2274223 in PLCE1 may serve as a potential biomarker for GC prognosis.

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2. METHODS

2.1. Ethics

The study was approved by the Lithuanian Bioethics Committee (Protocol No. 2/2008), Kaunas Regional Ethics Committee of Biomedical Surveys (Protocol no. BE-2-10), Central Medical Ethics Committee of Latvia (Protocol no. 01-29.1) and Ethics Committee of the Otto-von-Guericke Uni-versity Magdeburg (Protocols No. 63/08 and 34/08). The research was carried out in accordance with the Helsinki Declaration. All patients and controls gave their informed consent to take part in the study.

2.2. Design of the study: “Primary antibiotic resistance of H. pylori in a tertiary referral center in Lithuania” 2.2.1. Study population

Patients from Hospital of Lithuanian University of Health Sciences Kauno klinikos referred to upper gastrointestinal tract endoscopy (in outpa-tient clinic) due to dyspeptic symptoms, abdominal pain and (or) unclear cause anemia were included in the study. Patients who had previously received H. pylori eradication therapy or had been using proton pump inhi-bitors (PPI), antibiotics or bismuth compounds for the last 4-week period were excluded from the study. During the 3-year period (2013–2015) in total 242 adults and 55 children (<18 years old) were included (297 patients) in the study.

2.2.2. Biopsies

During upper endoscopy, two biopsies of gastric mucosa (one from

antrum and one from corpus) were obtained for culture of H. pylori. Biopsies

were transferred to tubes with serum bouillon – 10% glycerol and frozen immediately at –80°C until analysis.

2.2.3. Culture of H. pylori

All H. pylori detection tests (culture, susceptibility and genetic tests) were performed in H. pylori laboratory, Department of Clinical Microbio-logy, Rigshospitalet – Copenhagen University Hospital.

All biopsies were cultured on agar plates with 7% lysed and defibrinated horse blood (chocolate agar plates). The agar plates were incubated at 37°C in a microaerobic atmosphere (10% CO2, 5% O2 and 85% N2) up to 7 days.

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If there were no growth of H. pylori after 7 days, the culture was considered negative. In positive cultures colonies that macroscopically appeared as

H. pylori on the medium (small 0.5–2 mm size, round and convex colonies

that were transparent or brownish) were grown on a new chocolate agar plates. H. pylori was identified using following methods: microscopy (Gram-stained preparation from a culture. H. pylori appears as gram-negative curved rods), biochemistry (positive urease, catalase and oxidase tests) and MALDI-TOF-MS – matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (“Bruker MALDI Biotyper”). Each identified H. pylori strain was frozen and stored at –80°C for further analysis.

2.2.4. Susceptibility testing

Susceptibility of the H. pylori strains to amoxicillin, metronidazole, clarithromycin, ciprofloxacin, tetracycline and rifampicin was tested using E-tests (Biomerieux, France). H. pylori CCUG 17874 (ATCC 43504) was used as quality control (QC) strain. H. pylori suspensions were adjusted to McFarland (McF) turbidity standard of 4. The bacteria suspensions were spread on a chocolate agar plate and left to dry for 15 min before E-test strip was applied to each agar plate. The agar plates were incubated as described above. The results were read after 72 ± 1 h. Titer fold concentration break-points for H. pylori resistance to metronidazole was >8 mg/L; to clarithro-mycin >0.5 mg/L; to amoxicillin >0.125 mg/L, to tetracycline >1 mg/L and to rifampicin >1 mg/L according to the European Committee of Antibiotic Susceptibility Testing (EUCAST). The breakpoint for ciprofloxacin was settled as >1 mg/L corresponding to the EUCAST breakpoints for levofloxa-cin and the ciprofloxalevofloxa-cin breakpoint for many Gram-negative rods [124]. As

H. pylori is a slow growing microaerobic bacteria, the guidelines from

EUCAST had to be slightly modified as H. pylori needs a little higher ino-culum to grow sufficient within 48–72 h; therefore, McF 4 was used in this study and metronidazole susceptibility testing was adjusted in accordance with some inter- and intralaboratory variation which has been reported previously [125].

2.2.5. DNA extraction

To investigate whether H. pylori were present in the culture-negative biopsies, DNA extraction was performed on the 218 biopsies using the “DNeasy Blood & Tissue” (Qiagen, Germany) kit according to the manu-facturer’s instructions. Samples were stored at –20°C in the freezer until further analysis.

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Equipment:

1. Disposable gloves.

2. DNeasy Mini Spin Columns (colorless) in 2 mL Collection Tubes (Qiagen, Germany).

3. Microcentrifuge tubes (1.5 mL and 2 mL) – Eppendorf Tubes (Eppendorf AG, Germany).

4. Adjustable pipettes “Ependorf research” (Eppendorf AG, Germany). 5. Electronic pipette “Ependorf Xplorer” (Eppendorf AG, Germany). 6. Disposable tips for pipettes (Eppendorf AG, Germany).

7. Microcentrifuge with rotor for 1.5 mL and 2 mL tubes “Eppendorf Centrifuge 5810R” (Eppendorf AG, Germany).

8. Vortexer (Fisherbrand™ Variable Speed Mini Vortex Mixer, USA) 9. Thermomixer “Eppendorf Thermomixer comfort” (Eppendorf AG,

Germany).

Reagents:

1. Buffer ATL (50 mL bottle). 2. Buffer AL (54 mL bottle).

3. Buffer AW1 (concentrate, 95 mL bottle). 4. Buffer AW2 (concentrate, 66 mL bottle). 5. Buffer AE (2 × 60 mL bottles).

6. Proteinase K (6 mL bottle). 7. Ethanol 96% (Panreac, Spain).

DNA extraction from biopsies (DNeasy Blood & Tissue Kit) protocol:

•_{Buffer ATL and Buffer AL must be investigated for precipitation. If} there is precipitation, Buffers are heated at 56°C until the precipitation dissolves.

• Before use, to obtain a working solution, ethanol (96–100%) is added according to the bottle label to Buffer AW1 and Buffer AW2. •_{All centrifugation steps are carried out at room temperature (15–}

25°C) in a Micro centrifuge. All the centrifuging is at minimum 8000 rpm for 1 minute.

• Vortexing should be performed by pulse-vortexing for 5–10 s. • The thermomixer should be preheated to 56°C.

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1. 180 µL of Buffer ATL is added to 2 mL safe-lock Eppendorf tube. 2. Biopsy is added to the tube.

3. 20 µL of proteinase K to the tube.

4. The samples are placed in the preheated thermomixer and shacked. The samples are left in the thermomixer until biopsies dissolve. It takes about 1 hour and 30 min. Or the samples could be left overnight. 5. The samples are centrifuged briefly so that the droplets on the lid

would disappear.

6. 200 µL of Buffer AL is added to each sample and mixed by vortexing.

7. 200 µL ethanol (96–100%) is added to each sample and mixed. 8. The mixture is transferred to a mini spin column in a 2 mL collector

tube centrifuged. The collector tube is discarded.

9. The mini spin column is placed in a new collector tube and 500 µL of Buffer AW1, and centrifuged. The collector tube is discarded. 10. The mini spin column is placed in a new collector tube and 500 µL

of Buffer AW2 is added and centrifuged for 3 minutes at 14,000 rpm. The collector tube is discarded.

11. The mini spin column is placed in a 1.5 mL Eppendorf tube and 100 µL of Buffer AE is added directly on the membrane. The samp-les are incubated at room temperature for 2 minutes and then centri-fuged.

12. The mini spin column is discarded and the flow-through is saved. 13. The flow-through is stored at –20°C in the freezer until further

analysis.

2.2.6. Genotyping

1. Helicobacter genus-specific polymerase chain reaction (PCR) assay was conducted on purified DNA to investigate whether Helicobacter species were present (218 samples were analyzed).

2. The samples tested positive for Helicobacter spp. were further analyzed to find the H. pylori positives and to verify whether they were clarithromycin resistant (58 samples were analyzed).

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2.2.6.1. PCR

The polymerase chain reaction (PCR) method is used to amplify DNA in a defined region that is flanked by two primers [126]. PCR permits identi-fication of non-cultivatable or slow-growing microorganisms such as

H. pylori which are fastidious bacterial pathogens that are difficult to culture

by standard methods [127]. PCR method has been used in order to find

Heli-cobacter spp. in culture-negative samples.

Amplification reaction of bacterial 16S rDNA gene

The methods and conditions used for the PCR (for Genus-Level Identification of Helicobacter spp.) are described by Moyaert and colleagues. [128] (except that 2 µL of DNA template was used instead of 1 µL).

Helico-bacter genus-specific PCR primers, targeting 16S rDNA, are described by

Al-Soud and colleagues [127].

Equipment:

2. Adjustable pipettes (Eppendorf AG, Germany).

3. Electronic pipette “Ependorf Xplorer” (Eppendorf AG, Germany). 4. Disposable tips for pipettes (Eppendorf AG, Germany).

5. PCR tubes 0,2 mL “Eppendorf Fast PCR Tube Strips” (Eppendorf AG, Germany).

6. “Eppendorf Conical Tubes 15 mL” (Eppendorf AG, Germany). 7. Vortexer (Fisherbrand™ Variable Speed Mini Vortex Mixer, USA). 8. Thermal cycler “Biometra T-personal” (Biometra, Germany).

Reagents:

1. Sterile H2O (nuclease free water).

2. PCR Buffer (Promega, USA).

3. MgCl2 25 mM (Magnesium chloride) (Promega, USA).

4. GoTaq®_{DNA Polymerase (Promega, USA).}

5. dNTP Mix (Promega, USA).

6. 20 mM Forward primer 5’-CTA TGA CGG GTA TCC GGC-3’. 7. 20 mM Reverse primer 3’-CTC ACG ACA CGA GCT GAC- 5’.

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Table 2.2.6.1.1. Helicobacter genus-specific PCR primers, targeting 16S rDNA1

Primer2 _{Primer sequence} _{PCR product size (bp)}

Forward (254–271) 5’-CTA TGA CGG GTA TCC GGC-3’ ~780 Reverse (1018–1035) 3’-CTC ACG ACA CGA GCT GAC-5’

1_{Primers according to Al-Soud and colleagues [127].}2_{Primer positions are based on the}

sequence of Escherichia coli 16S rDNA.

Table 2.2.6.1.2. PCR protocol

Reagents Final concentration Total volume 25 µL (for 1 reaction)

10×PCR Buffer – 2,5 µL

MgCl2 25 mM 5 µL

dNTP 200 µM 5 µL

Forward primer 20 mM 0,25 µL

Reverse primer 20 mM 0,25 µL

Taq polymerase 5 U/µL 0,2 µL

Sterile H2O 13,3 µL

Template DNA 2 µL

Method:

1. The master mix is made for 50 reactions according to the protocol (Table 2.2.6.1.2): 125 µL 10×PCR Buffer, 250 µL 25 mM MgCl2,

250 µL 200 µM dNTP, 12,5 µL 20 mM forward primer, 12,5 µL 20 mM reverse primer, 665 µL sterile H2O and finally 10 µL 5 U/µL

Taq polymerase is added (the master mix is briefly vortexed).

2. 0.2 mL PCR stripes are put on ice. 25 µL of master mix is put in each test tube with a pipette.

3. 2 µl of extracted (template) DNA is pipetted to each test tube. 4. Negative control: no DNA is added, positive control: the genomic DNA

of H. pylori CCUG 17874 (ATCC 43504) is added to the test tube. 5. The PCR stipes are put in the thermal cycler “Biometra T-personal”.

Amplification conditions: 1) 94°C for 5 minutes. 2) 35 cycles of: •_{94°C for 30 seconds.} •_{55°C for 1 minute.} •_{72°C for 90 seconds.} 3) 72°C for 5 minutes. 4) 4°C pause.

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2.2.6.2. Agarose gel electrophoresis

In order to detect amplicons (amplification products), gel electrophoresis was used. It is a procedure for separating DNA by size for visualization. An electrical field is used to move the negatively charged DNA through an agarose gel matrix toward a positive electrode during electrophoresis. Shorter DNA fragments migrate through the gel more quickly than longer ones. Therefore, the approximate length of a DNA fragment can be determined by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths) [126].

Reagents:

1. Precast 1.5% agarose gel “1.5% Agarose TAE with EtBr” (EmbiTec, USA).

2. Electrophoresis buffer: 1 X TAE Buffer Running Buffer (EmbiTec, USA).

3. 100 bp DNA Ladder (Promega, USA).

4. “The Blue/Orange Loading Dye, 6X” Promega, USA).

Equipment:

1. Electrophoresis system “RunOne™ Electrophoresis System 25– 100 V” with integrated power supply (EmbiTec, USA).

2. Adjustable pipettes (Eppendorf AG, Germany). 3. Disposable gloves.

4. Disposable tips for pipettes (Eppendorf AG, Germany).

5. Gel documentation system “Bio-Rad ChemiDoc XRS Molecular Chemiluminescence Fluorescence Gel Documentation” (Bio-Rad, USA).

Method:

1. Precast 1.5% agarose gel with the tray is placed on the running platform of the RunOne tank. The agarose gel should be oriented so that the wells would be closer to the Power Supply.

2. About 250 to 300 mL of the matching running buffer is poured into the tank. The agarose gel should be just covered with the running buffer. The buffer must not exceed the maximum line marked on the internal sides of the tank.

3. The wells should be flushed with the running buffer to remove any debris.

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4. The wells should be checked for air bubbles. If any found – they must be removed with a pipette.

5. 10 µL of PCR product is mixed with 2 µL loading buffer “The Blue/Orange Loading Dye, 6X”.

6. The samples are loaded into the wells.

7. 100 bp DNA Ladder is used as a molecular size marker (it is loaded into the first well).

8. Electrophoresis lasts for 30 minutes at 100 V.

9. PCR products are visualized using Bio-Rad gel documentation system.

Fig. 2.2.6.2.1. Amplification products of 16S rDNA gene

enable identification of Helicobacter spp.

2.2.6.3. RT – PCR

A real-time polymerase chain reaction (RT – PCR) is a laboratory techni-que of molecular biology based on PCR. It monitors the amplification of a targeted DNA molecule during the PCR in “real-time”, and not at its end, as in conventional PCR. A specific DNA sequence of the pathogen’s genome is amplified and the generated PCR-product is detected by an oligonucleotide-probe labelled with a fluorescent dye [126].

To find the H. pylori positives and to verify if they were clarithromycin resistant, the samples positive for presence of Helicobacter spp. were further analyzed using H. pylori ClariRes Assay (Ingenetix GmbH, Vienna, Austria) which was used according to the manufacturer’s directions. In order to

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evaluate the compatibility between the two methods (culture and ClariRes assay), randomly 10 clarithromycin susceptible H. pylori strains and 10 clari-thromycin resistant H. pylori strains were chosen from the culture positive samples and analyzed with ClariRes Assay.

The H. pylori ClariRes Assay is a test for the simultaneous detection of

H. pylori and of the three major point mutations in the 23S rRNA gene

associated with clarithromycin resistance in H. pylori isolates. The three point mutations and the wild-type-sequence of the 23S rRNA gene of H. pylori are amplified in one PCR reaction and are subsequently differentiated by melting curve analyses in a LightCycler®_[129].

Reagents:

1. Primers and probe for H. pylori and internal positive control (IC) detection 25 µL (H. pylori ClariRes Assay Mix).

2. Internal positive control 25 µL (H. pylori ClariRes Internal Positive Control).

3. Water as negative control 1000 µL (ClariRes Water (PCR grade)). 4. Control-DNA of a H. pylori 23S rRNA wild-type (clarithromycin

susceptible) 25 µL (H. pylori Wild-type Positive Control).

5. Control-DNA of a H. pylori 23S rRNA mutant (A2142G) (clarithro-mycin resistant) 25 µL (H. pylori Resistant Positive Control). 6. LCTM_{-FastStart DNA Master SYBR}®_{Green I (Roche Diagnostics,}

Switzerland).

7. MgCl2 25 mM (Magnesium chloride) (Promega, USA). Equipment:

2. Adjustable pipettes (Eppendorf AG, Germany).

3. Electronic pipette “Ependorf Xplorer” (Eppendorf AG, Germany). 4. Disposable tips for pipettes (Eppendorf AG, Germany).

5. Vortexer (Fisherbrand™ Variable Speed Mini Vortex Mixer, USA). 6. Desktop centrifuge with rotor for 2 mL reaction tubes “Eppendorf

Centrifuge 5810 R”.

7. Capillaries (20 μL) LightCycler® (Roche Diagnostics, Switzerland). 8. Cooling Block LightCycler® (Roche Diagnostics, Switzerland). 9. Capping Tool LightCycler®_{(Roche Diagnostics, Switzerland).}

10. LightCycler®_{2.0 Instrument – Real-Time PCR System (Roche}

Diag-nostics, Switzerland).

11. Carousel Centrifuge LightCycler®_{(Roche Diagnostics,}

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Table 2.2.6.3.1. The master mix preparation protocol

Reagents Total volume 18 µL (for 1 reaction)

H2O (nuclease free water) 12.1 µL

MgCl2 25 mM 2.4 µL

LCTM_{-FastStart DNA Master SYBR}®_{Green I} _{2.0 µL}

H. pylori ClariRes Assay Mix 0.5 µL

H. pylori ClariRes Internal Positive Control;

freshly diluted 1:100 1.0 µL

Table 2.2.6.3.2. LightCycler®_{PCR protocol}

Step Temperature Incubation time Cycle(s) Function

1 95°C 10 min. 1 Denaturation 2 95°C 5 sec. 70 Amplification 65°C 10 sec. 72°C 6 sec. 3 95°C 0 sec. 1 Melting 37°C 1 min. 95°C 0 sec. 4 40°C 10 sec. 1 Cooling Method:

1. Before use all ingredients are allowed to thaw in a room temperature. 2. Internal positive control is diluted freshly 1:100 with provided water (ClariRes Water). The tube is gently flicked for a few times in order to mix.

3. The master mix is prepared from the ingredients listed in Table 2.2.6.3.1.

4. 18 μL of the master mix is pipetted into the reservoir of each capillary.

5. 2 μL of template DNA is added.

6. 2 μL of nuclease free water is added to the negative control capillary. 7. 1 μL of each positive control (1 × wild-type, 1 × resistant – control A2141G) is added into the capillary (positive controls are added as a final step in order to avoid contamination of the samples).

8. The capillaries are closed and briefly centrifuged.

9. The capillaries are placed into LightCycler®_{2.0 Instrument}

(Real-Time PCR System). The gene amplification is carried out according to PCR protocol presented in Table 2.2.6.3.2.

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The PCR-results were analyzed after choosing fluorescence display option 670 nm. The presence of melting curves also was checked manually. Example of the results are shown in Fig. 2.2.6.3.1.

Fig. 2.2.6.3.1. Example of melting curves of H. pylori clarithromycin

resistant (mutant) or clarithromycin susceptible (wild-type) strains and of the internal positive control (IC) [129].

2.2.7. Outpatient antibiotic consumption in Lithuania

Data about all authorized and available on market macrolides (erythro-mycin, spira(erythro-mycin, azithromycin and clarithromycin), lincosamides (clinda-mycin), amoxicillin, metronidazole and ciprofloxacin in Lithuania during the period 2003–2015 were evaluated using WHO ATC DDD (World Health Organization – Anatomical Therapeutic Chemical – Defined Daily Dose) drug utilization methodology as described in literature [56, 130]. Total drug utilization volume was calculated and expressed in defined daily dose (DDD)/1000 inhabitants/day utilization units. The volume of antibiotics use in pediatric population was expressed in DDD/1000 children/day utilization units. Data about population were obtained from the official national statistics website. Data on sales of antibiotics in Lithuania were obtained from a country-specific database (www.softdent.lt), which comprises all purchases of prescription and over-the-counter medicines from wholesalers and retailers in Lithuania. The data on sales were retrieved as units of antibiotics packages. All pharmaceutical forms available on market during the study period were selected for calculations, and calculated drug utilization volume was 100% (DU 100%).

Among the currently available macrolides, only clarithromycin is widely used to treat H. pylori infection. There is well-documented evidence for cross-resistance to macrolides and clarithromycin resistance may originate from the previous consumption of macrolides to treat other infectious diseases

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[20]. Therefore, data about all available on market macrolides rather than only clarithromycin was evaluated.

Although macrolide and lincosamide antibiotics are chemically distinct but they share a similar mode of action and there is cross resistance to macrolides and lincosamides [131]. Hence it was necessary to access the data about clindamycin utilization.

2.2.8. Statistical analysis

The data were processed using Microsoft Excel (2013) and SPSS 20.0 software (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp). Differences between the groups were assessed with chi-square, Fisher’s exact and Z tests when appropriate. 95% confidence intervals (CI) were calculated for antibiotic resistance rates where appropriate. A p-value of <0.05 was considered statistically significant.

2.3. Design of the study: “Genetic host predisposition to H. pylori infection and related diseases – atrophic gastritis and gastric cancer”

2.3.1. Study population

Patients and controls were recruited during 2005–2017 at three gastro-enterology centers in Lithuania (Department of Gastrogastro-enterology, Hospital of Lithuanian University of Health Sciences Kauno klinikos), Latvia (Riga East University Hospital) and Germany (Department of Gastroenterology, Hepa-tology and Infectious Diseases, Otto-von-Guericke University, Magdeburg). All patients in the control and atrophic gastritis (AG) groups underwent upper endoscopy with biopsies due to dyspeptic symptoms but had no history of malignancy. Patients without atrophy or intestinal metaplasia on gastric biopsies based on the Sydney classification [132] were included in the control group, while patients with premalignant conditions constituted the AG group. This consisted of patients with high-risk atrophic gastritis (HRAG), defined as pan-gastritis (similar inflammatory scores in antrum and corpus), corpus-predominant gastritis with or without the presence of gastric atrophy, and intestinal metaplasia either in the antrum or corpus of the stomach [133]. All GC patients had histopathological documentation of gastric adenocarcinoma. The inclusion criterion for the study population was no previous history of

H. pylori eradication. H. pylori status was determined by testing for anti-H. pylori IgG antibodies in serum using the ELISA method. The results were

interpreted according to the locally established titers. All patients in the present study were of European descent.

CHARACTERISTICS OF HELICOBACTER PYLORI INFECTION: RESISTANCE TO ANTIBIOTICS AND ASSOCIATION WITH HOST TLR1, PRKAA1, PSCA, MUC1 AND PLCE1 GENE POLYMORPHISMS

Gintarė Dargienė