**5. Treatment of** *H. pylori* **infection**

of *H. pylori* infections in patients with gastric cancer when other tests are negative [21]. Due to local strain distribution of *H. pylori,* the serology kits should be made by using local *H. pylori*

Gastrin and pepsinogen are compounds produced in the stomach that depend on the changes in the gastric mucosa, and the serum levels of pepsinogens are a marker of atrophic gastritis [22]. This can be combined with the *H. pylori* antibody test to predict the risk of developing

Molecular methods have been of increasing interest in the field of microbiology and for detection of *H. pylori*. Polymerase chain reaction (PCR) seems to be more sensitive than any other method to detect *H. pylori* [23]. The main problem is that the method does not distinguish between live bacteria and DNA from dead bacteria. Real-time PCR (RT-PCR), which is a fast and quantitative PCR, seems to be more sensitive than classical PCR [24]. By sequencing the 16S RNA or 23S RNA region, it is possible to detect *Helicobacter* species and susceptibility to clarithromycin and tetracycline [25–27]. However, it is a more expensive and time-consuming method. A commercial kit has combined detection of *H. pylori* and susceptibility to clarithromycin in a classical PCR. However, culture is still needed for a full susceptibility testing. There are so many point mutations causing resistance to antibiotics in *H. pylori* that a full

During the last decade, an increased number of *H. pylori* have become resistant to antibiotics, especially to clarithromycin and levofloxacin [29]. The resistance rates to metronidazole have always been more than 15% worldwide, but the increasing resistance rates to clarithromycin and levofloxacin in some areas have become higher than 10–15%. Thus, these antibiotics are not recommended for first-line therapy of *H. pylori* without prior susceptibility testing [21]. It is common to treat *H. pylori* infections without prior susceptibility testing, and different studies show a much lower resistance rate to clarithromycin in *H. pylori* from untreated patients than in *H. pylori* from previously treated patients [30–32]. It is therefore of the greatest impor-

The susceptibility testing of *H. pylori* can be done by various methods. The most common are

The dilution method is regarded to be the golden standard for susceptibility testing. A twofold dilution row of the test antibiotic is made. A standard number of bacteria (McFarland 3) are added to each tube with antibiotics. The bacterial growth is inhibited by high concentrations of antibiotics. The first tube with bacterial growth is called the minimal inhibitory concentration (MIC). *H. pylori* should be grown for 48–72 hours under microaerobic conditions. It may be difficult to find a suitable media in which *H. pylori* grows fast enough, and the slightest contamination will grow faster than *H. pylori* and thereby spoil the susceptibility testing. The disk diffusion test requires a small tablet of an antibiotic. The tablet is placed on the agar plate and is incubated for 3 days. After 3 days, there will be a zone around the tablet with no

susceptibility analysis can only be detected by whole genome sequencing [28].

tance to make susceptibility testing after the first treatment failure.

strains, and the kits should be locally validated [21].

96 Helicobacter Pylori - New Approaches of an Old Human Microorganism

**4.** *H. pylori* **susceptibility to antibiotics**

dilution methods, disk diffusion, and E-test.

gastric cancer.

*H. pylori* infections are usually treated with a combination of antibiotics and nonantibiotics (proton pump inhibitor [PPI] or bismuth salts). Usually, a combination of two or three antibiotics is used, as the effect of monotherapy has been found insufficient. The most commonly used antibiotics are amoxicillin, clarithromycin, metronidazole, fluoroquinolones, tetracycline, and rifampicin (**Table 3**).

*H. pylori* is found in very different environments such as the gastric lumen with a relatively low pH, in between the epithelial cells and on the basement membrane with a neutral pH but protected as intracellular microorganisms. When choosing antibiotics, it is important to select antibiotic to which *H. pylori* is sensitive and is active in all the environmental niches where *H. pylori* occurs. It is also important to look at the duration of the efficacy of antibiotics to keep stable levels above the minimal inhibitory concentrations.

PPI in standard doses do not have antibacterial effect on *H. pylori*, but 5–10 times higher doses have a direct effect on *H. pylori*. Bismuth salts binds to the surface of *H. pylori* but have


A large multinational study tested *H. pylori* resistance in 18 European countries [29]. All 18 countries used E-test for the susceptibility testing and only tested patients who had never been treated for *H. pylori* before. In total, 2204 people were included in the study, and the resistance rate for adults were 18% for clarithromycin, 14% for levofloxacin, and 35% for metronidazole. They found a significant association between the use of only long-acting macrolides and clarithromycin resistance. The levofloxacin resistance was significantly associated

Gastric Microbiota and Resistance to Antibiotics http://dx.doi.org/10.5772/intechopen.80662

The prevalence of *H. pylori* resistance to antibiotics was tested in Denmark in 1997, 1998–2004, and 2013 [71–73]. Throughout the years, the resistance for clarithromycin has increased from 0% in 1997 to 53% in 2013, and likewise, the resistance for metronidazole increased from 20 to 74% [12–14]. None of the studies mention whether or not the patients have had *H. pylori* eradication therapy prior to testing or not, which might explain the huge increase in resistance.

International guidelines recommend first line of treatment of *H. pylori* infections with 10 days of triple therapy (PPI, clarithromycin, and metronidazole or amoxicillin). If this fails, a treatment with four types of medicine (PPI, bismuth subsalicylate, tetracycline, and metronidazole) for 2 weeks is recommended. After treatment failure for the second time, it is recommended to

The primary and secondary resistance rate for *H. pylori* has only been described in eight studies [30, 32, 40, 43, 58, 65, 66, 74]. By using "Review Manager 5.3," it is possible to compare the studies via Forest plots. The meta-analyses show that the secondary resistance is significantly

The meta-analysis shows a high increasing resistance rate for all three antibiotics when the patient had been treated for *H. pylori* previously. The high and increasing resistance rates to metronidazole, clarithromycin, and levofloxacin make it uncertain that these antibiotics should be recommended as the first-line therapy of *H. pylori* infections without prior endos-

Another way to overcome *H. pylori* infections is with a vaccine. In the past couple of years, many studies have investigated developing an effective and safe vaccine. The development of an effective vaccine is complicated by the noninvasive nature of *H. pylori.* It stays in the lumen of the stomach and does not cross the epithelium. Therefore, the vaccine should affect T helper memory cells, which are required to stay in the lumen during a *H. pylori*

Appropriate bacterial antigens, safe and effective adjuvants, and a route of delivery are required for developing a vaccine. For the bacterial antigen, most studies use urease, but other antigens are investigated for example Cag L. The CagL is a protein essential for the pathogenesis of *H. pylori*. It binds to integrins in the mucosa and triggers the release of the carcinogen CagA to the host cells

**6.1. Effect of antibiotic treatment on** *H. pylori* **resistance rates**

perform a gastroscopy and susceptibility testing for *H. pylori* [21].

with the use of quinolone.

higher (p < 0.001) than the primary.

**6.2. Vaccine**

infection [75].

copy and susceptibility testing (**Figure 4A**–**C**).

**Table 3.** Commonly used antibiotics and nonantibiotics for treatment of *H. pylori* infections.

a relatively little antibacterial effect. However, bismuth salts affect the respiratory chain at the same points as metronidazole and thereby reverts metronidazole resistance in *H. pylori* and thus becomes sensitive to metronidazole.

#### **6. Prevalence of** *H. pylori* **resistance to antibiotics**

When analyzing different studies around the world, the primary resistance rate for *H. pylori* varies. The highest rate of primary metronidazole (MTZ) resistance is found in Africa (52%) followed by South America (49%) and Asia (43%). The lowest resistance rate is found in Europe (35%). The highest primary resistance rates for clarithromycin and levofloxacin are found in South America (20 and 27%) while the lowest rates are found in Europe (12 and 10%) [30–32, 34–67]. There is a significantly (p < 0.001) higher risk of primary metronidazole and levofloxacin resistance in Asian when compared to Europe.

The high rate of metronidazole resistance seen in developing countries may be due to the high use of metronidazole for treatment of parasites and gynecological infections [62, 68]. It is therefore likely that the patients who are treated for *H. pylori* with metronidazole for the first time are resistant for this treatment. It is recommended to use bismuth therapy together with metronidazole in the first-line treatment in areas with high metronidazole resistance [21].

The high resistance rates for clarithromycin and levofloxacin in South America, Africa, and Asia can be due to the use of huge amounts of antibiotics in general [69]. Typically, the diagnostics are not precise, and the patients are treated with more a broad spectrum of antibiotics for a longer period. This can lead to a faster development of resistance in *H. pylori* [70].

A large multinational study tested *H. pylori* resistance in 18 European countries [29]. All 18 countries used E-test for the susceptibility testing and only tested patients who had never been treated for *H. pylori* before. In total, 2204 people were included in the study, and the resistance rate for adults were 18% for clarithromycin, 14% for levofloxacin, and 35% for metronidazole. They found a significant association between the use of only long-acting macrolides and clarithromycin resistance. The levofloxacin resistance was significantly associated with the use of quinolone.

The prevalence of *H. pylori* resistance to antibiotics was tested in Denmark in 1997, 1998–2004, and 2013 [71–73]. Throughout the years, the resistance for clarithromycin has increased from 0% in 1997 to 53% in 2013, and likewise, the resistance for metronidazole increased from 20 to 74% [12–14]. None of the studies mention whether or not the patients have had *H. pylori* eradication therapy prior to testing or not, which might explain the huge increase in resistance.

#### **6.1. Effect of antibiotic treatment on** *H. pylori* **resistance rates**

International guidelines recommend first line of treatment of *H. pylori* infections with 10 days of triple therapy (PPI, clarithromycin, and metronidazole or amoxicillin). If this fails, a treatment with four types of medicine (PPI, bismuth subsalicylate, tetracycline, and metronidazole) for 2 weeks is recommended. After treatment failure for the second time, it is recommended to perform a gastroscopy and susceptibility testing for *H. pylori* [21].

The primary and secondary resistance rate for *H. pylori* has only been described in eight studies [30, 32, 40, 43, 58, 65, 66, 74]. By using "Review Manager 5.3," it is possible to compare the studies via Forest plots. The meta-analyses show that the secondary resistance is significantly higher (p < 0.001) than the primary.

The meta-analysis shows a high increasing resistance rate for all three antibiotics when the patient had been treated for *H. pylori* previously. The high and increasing resistance rates to metronidazole, clarithromycin, and levofloxacin make it uncertain that these antibiotics should be recommended as the first-line therapy of *H. pylori* infections without prior endoscopy and susceptibility testing (**Figure 4A**–**C**).

#### **6.2. Vaccine**

a relatively little antibacterial effect. However, bismuth salts affect the respiratory chain at the same points as metronidazole and thereby reverts metronidazole resistance in *H. pylori* and

H2 blocker

Clarithromycin Metronidazole Tetracycline Levofloxacin Ciprofloxacin Rifampicin

Bismuth nitrate Bismuth citrate Bismuth subsalicylate

When analyzing different studies around the world, the primary resistance rate for *H. pylori* varies. The highest rate of primary metronidazole (MTZ) resistance is found in Africa (52%) followed by South America (49%) and Asia (43%). The lowest resistance rate is found in Europe (35%). The highest primary resistance rates for clarithromycin and levofloxacin are found in South America (20 and 27%) while the lowest rates are found in Europe (12 and 10%) [30–32, 34–67]. There is a significantly (p < 0.001) higher risk of primary metronidazole and

The high rate of metronidazole resistance seen in developing countries may be due to the high use of metronidazole for treatment of parasites and gynecological infections [62, 68]. It is therefore likely that the patients who are treated for *H. pylori* with metronidazole for the first time are resistant for this treatment. It is recommended to use bismuth therapy together with metronidazole in the first-line treatment in areas with high metronidazole

The high resistance rates for clarithromycin and levofloxacin in South America, Africa, and Asia can be due to the use of huge amounts of antibiotics in general [69]. Typically, the diagnostics are not precise, and the patients are treated with more a broad spectrum of antibiotics for a longer period. This can lead to a faster development of resistance in *H. pylori* [70].

thus becomes sensitive to metronidazole.

resistance [21].

**6. Prevalence of** *H. pylori* **resistance to antibiotics**

**Table 3.** Commonly used antibiotics and nonantibiotics for treatment of *H. pylori* infections.

**Group Preparation** Antibiotics Amoxicillin

98 Helicobacter Pylori - New Approaches of an Old Human Microorganism

Nonantibiotics PPI

levofloxacin resistance in Asian when compared to Europe.

Another way to overcome *H. pylori* infections is with a vaccine. In the past couple of years, many studies have investigated developing an effective and safe vaccine. The development of an effective vaccine is complicated by the noninvasive nature of *H. pylori.* It stays in the lumen of the stomach and does not cross the epithelium. Therefore, the vaccine should affect T helper memory cells, which are required to stay in the lumen during a *H. pylori* infection [75].

Appropriate bacterial antigens, safe and effective adjuvants, and a route of delivery are required for developing a vaccine. For the bacterial antigen, most studies use urease, but other antigens are investigated for example Cag L. The CagL is a protein essential for the pathogenesis of *H. pylori*. It binds to integrins in the mucosa and triggers the release of the carcinogen CagA to the host cells


The routes of delivery that have been tested are sublingual, intranasal, respiratory, and oral [79]. A study on humans from China (2015) tested a vaccine based on a urease B subunit and heat-labile enterotoxin B subunit (gene derived from *E. coli* H44815) [80]. The vaccine was taken orally three times (day 0, 14, and 28). This study showed a vaccine efficacy of 71.8% in the first year, 55% in in the second year, and 55.8% in the third year after vaccinations. Even though these findings are excellent, a 100% effective vaccine is still not developed. More studies and longer time follow-ups are needed before a fully effective vaccine is on the market. If a fully effective vaccine is made, it would be the best heath measure against *H. pylori* infections

Gastric Microbiota and Resistance to Antibiotics http://dx.doi.org/10.5772/intechopen.80662

The human gastric microbiota may be difficult to estimate since samples for microbiome investigations often are contaminated with oral bacterial flora during gastroscopy. And the studies in these fields do not make any attempt to remove the oral contamination prior to sequencing. Histological examination of biopsies reveals *H. pylori* as the only bacteria in close relation to the epithelial cells in the gastric mucosa. When *H. pylori* is seen in stomach samples, there is always a strong humoral and cellular immune response to *H. pylori* and it thereby fulfills the criteria for

Thus, in noncancer patients, *H. pylori* seems to be the gastric microbiota. In patients with gastric cancer, there may be a different situation as the mucosa is disintegrated and an overgrowth of intestinal bacteria is common. However, it remains to be shown that the intestinal bacteria adhere to the gastric mucosa and cause a local immune response. It is, therefore,

An increasing resistance to antibiotics in *H. pylori* has been seen worldwide especially to metronidazole, clarithromycin, and levofloxacin. This is a worrying development as it may interfere with our recommendations for primary treatment of *H. pylori* without susceptibility testing. It is a question how fast the resistance occurs. Should susceptibility testing be done after first treatment failure or can it wait until the second treatment failure as recommended? At least the resistance rates are much higher in previously treated patients than in untreated patients. Due to the high resistant rates, it is necessary to perform a susceptibility test before starting the treatment. The advantages would be a better and maybe quicker eradication of the *H. pylori* infection. Disadvantages of early susceptibility testing are the cost and time of the analyses. Biopsies are an invasive method and may often be painful for the patient. Furthermore, it takes up to 14 days before a full susceptibility test is completed, so the real treatment starts approximately 2 weeks after the doctor confirms the presence of *H. pylori*. By this time, the patient could have been done with the first line of treatment. In the short perspective, a quick susceptibility test would be very time consuming, but in the long perspective, it might save the patient from several treatments and prevent the relapse of the *H. pylori* infection. But it also gives a better overview on how quickly *H. pylori* develops resistance to the recommended treatment. When detecting *H. pylori,* the best would be a quick a method that was as quick as PCR but also made it possible to have a full susceptibility test incorporated. New primers for detecting

a true infection but also a colonization. This has not been shown for any other bacteria.

believed that *H. pylori* is still the most important gastric pathogen.

and gastric cancer.

**7. Discussion**



**Figure 4.** Meta-analysis for MTZ (A), CLR (B), and LEV (C). For all three antibiotics, there is a higher odds ratio for resistance if the patient is previously treated for infection with *H. pylori*.

through the type IV secretin system. CagL also introduces an IL-8 response, which causes inflammation [76]. The use of CagL in a subunit vaccine was investigated by Choudhari et al. in 2013 [75]. The study showed that CagL was stable in pH 4–6 and that sucrose enhances the stability.

The use of heat shock proteins in a vaccine introduced protective immunity without requiring the addition of an adjuvant. The protection, however, is not optimal because sterilizing immunity is not obtained, which is shown in a study from 2014 [77].

A derivate of the cholera toxin (CTA1-DD) and safe nontoxic mutants of *Escherichia coli* heat labile toxin (dm2T) have also been tested as potential adjuvants. CTA1-DD enhances the Th1 and Th17 immunity and reduces the bacterial colonization by three- to eight-fold [78]. The use of dm2T was equally as effective as the gold standard *H. pylori* vaccine containing cholera toxin [79].

The routes of delivery that have been tested are sublingual, intranasal, respiratory, and oral [79]. A study on humans from China (2015) tested a vaccine based on a urease B subunit and heat-labile enterotoxin B subunit (gene derived from *E. coli* H44815) [80]. The vaccine was taken orally three times (day 0, 14, and 28). This study showed a vaccine efficacy of 71.8% in the first year, 55% in in the second year, and 55.8% in the third year after vaccinations. Even though these findings are excellent, a 100% effective vaccine is still not developed. More studies and longer time follow-ups are needed before a fully effective vaccine is on the market. If a fully effective vaccine is made, it would be the best heath measure against *H. pylori* infections and gastric cancer.

#### **7. Discussion**

through the type IV secretin system. CagL also introduces an IL-8 response, which causes inflammation [76]. The use of CagL in a subunit vaccine was investigated by Choudhari et al. in 2013 [75]. The study showed that CagL was stable in pH 4–6 and that sucrose enhances the stability. The use of heat shock proteins in a vaccine introduced protective immunity without requiring the addition of an adjuvant. The protection, however, is not optimal because sterilizing

**Figure 4.** Meta-analysis for MTZ (A), CLR (B), and LEV (C). For all three antibiotics, there is a higher odds ratio for

A derivate of the cholera toxin (CTA1-DD) and safe nontoxic mutants of *Escherichia coli* heat labile toxin (dm2T) have also been tested as potential adjuvants. CTA1-DD enhances the Th1 and Th17 immunity and reduces the bacterial colonization by three- to eight-fold [78]. The use of dm2T was equally as effective as the gold standard *H. pylori* vaccine containing cholera toxin [79].

immunity is not obtained, which is shown in a study from 2014 [77].

resistance if the patient is previously treated for infection with *H. pylori*.

100 Helicobacter Pylori - New Approaches of an Old Human Microorganism

The human gastric microbiota may be difficult to estimate since samples for microbiome investigations often are contaminated with oral bacterial flora during gastroscopy. And the studies in these fields do not make any attempt to remove the oral contamination prior to sequencing. Histological examination of biopsies reveals *H. pylori* as the only bacteria in close relation to the epithelial cells in the gastric mucosa. When *H. pylori* is seen in stomach samples, there is always a strong humoral and cellular immune response to *H. pylori* and it thereby fulfills the criteria for a true infection but also a colonization. This has not been shown for any other bacteria.

Thus, in noncancer patients, *H. pylori* seems to be the gastric microbiota. In patients with gastric cancer, there may be a different situation as the mucosa is disintegrated and an overgrowth of intestinal bacteria is common. However, it remains to be shown that the intestinal bacteria adhere to the gastric mucosa and cause a local immune response. It is, therefore, believed that *H. pylori* is still the most important gastric pathogen.

An increasing resistance to antibiotics in *H. pylori* has been seen worldwide especially to metronidazole, clarithromycin, and levofloxacin. This is a worrying development as it may interfere with our recommendations for primary treatment of *H. pylori* without susceptibility testing. It is a question how fast the resistance occurs. Should susceptibility testing be done after first treatment failure or can it wait until the second treatment failure as recommended? At least the resistance rates are much higher in previously treated patients than in untreated patients.

Due to the high resistant rates, it is necessary to perform a susceptibility test before starting the treatment. The advantages would be a better and maybe quicker eradication of the *H. pylori* infection. Disadvantages of early susceptibility testing are the cost and time of the analyses. Biopsies are an invasive method and may often be painful for the patient. Furthermore, it takes up to 14 days before a full susceptibility test is completed, so the real treatment starts approximately 2 weeks after the doctor confirms the presence of *H. pylori*. By this time, the patient could have been done with the first line of treatment. In the short perspective, a quick susceptibility test would be very time consuming, but in the long perspective, it might save the patient from several treatments and prevent the relapse of the *H. pylori* infection. But it also gives a better overview on how quickly *H. pylori* develops resistance to the recommended treatment.

When detecting *H. pylori,* the best would be a quick a method that was as quick as PCR but also made it possible to have a full susceptibility test incorporated. New primers for detecting antibiotic resistance are in progress, but the problem is that there are many different mutations leading to the same resistance profile. *H. pylori* only develops antibiotic resistance by mutation in the genome. For MTZ, mutations in at least nine different genes are known to contribute to MTZ resistance [13]. If the detecting of MTZ resistance should be made by PCR, it would be necessary to perform the PCR with many different primers all looking for one specific mutation. In theory, this would be the most sensitive way to find MTZ resistance, but in practice, it would be almost impossible, take a lot of time, and would be expensive.

**References**

2012;**70**(Suppl 1):S38-S44

2011;**5**(4):574-579

Washington DC: ASM Press; 2015

[1] Ursell LK, Metcalf JL, Parfrey LW, et al. Defining the human microbiome. Nutrition Reviews.

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[2] Jackson MA, Bonder MJ, Kuncheva Z, et al. Detection of stable community structures within gut microbiota co-occurrence networks from different human populations. PeerJ. 2018;**6** [3] Jorgensen JH, Pfaller MA, Karen C, et al. Manual of Clinical Microbiology. 11th ed.

[4] Aviles-Jimenez F, Vazquez-Jimenez F, Medrano-Guzman R, et al. Stomach microbiota composition varies between patients with non-atrophic gastritis and patients with intes-

[5] Wang L, Zhou J, Xin Y, Geng C, et al. Bacterial overgrowth and diversification of microbiota in gastric cancer. European Journal of Gastroenterology & Hepatology. 2016;**28**(3):261-266

[6] Bik EM, Eckburg PB, Gill SR, et al. Molecular analysis of the bacterial microbiota in the human stomach. Proceedings of the National Academy of Sciences. 2006;**103**(3):732-737

[7] Maldonado-Contreras A, Goldfarb KC, Godoy-Vitorino F, et al. Structure of the human gastric bacterial community in relation to *Helicobacter pylori* status. The ISME Journal.

[8] Yang I, Nell S, Suerbaum S. Survival in hostile territory: the microbiota of the stomach.

[9] Liu X, Nie W, Liang J, et al. Interaction of *Helicobacter Pylori* with other microbiota species in the development of gastric cancer. Archives of Clinical Microbiology. 2017;**8**(2) [10] Carroll AC, Wong A. Plasmid persistence: Costs, benefits and the plasmid paradox.

[11] Dorward DW, Garon CF. DNA-binding proteins in cells and membrane blebs of *Neisseria* 

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Due to the enormous amount of mutations leading to antibiotic resistant, the culture and susceptibility testing done by E-test is still the best and most economical way.

The increasing resistant rates to the most commonly used antibiotics raises the question of whether other antibiotics or combinations of antibiotic and nonantibiotic should be used for primary treatment of *H. pylori* infections without susceptibility testing. Bismuth compounds in standard doses, proton pump inhibitors, and acid suppressing compounds in high doses may convert the MTZ resistance [81]. This makes MTZ useful in combination with these compounds, especially the bismuth compounds, which have been shown in clinical studies [21]. Nonantibiotics such as neuroleptics and other compounds acting on the central nerves system have anti–*H. pylori* effect *in vitro* [82] and compounds without effect on the central nervous system may be candidates for alternative treatment. Herbs like broccoli and green tee have some effect on *H. pylori* and may in combination with antibiotics and nonantibiotics be candidates for treatment in the future [83].

### **8. Conclusion**

*H. pylori* is the most important gastric pathogen and may constitute the true gastric microbiota. It is, therefore, important to follow the development of resistance in *H. pylori* to antibiotics. With the increased resistance of *H. pylori* to metronidazole, clarithromycin, and levofloxacin, it may be doubtful if these antibiotics can be recommended as primary treatment without susceptibility testing.

#### **Conflict of interest**

The authors declare no conflicts of interests.

#### **Author details**

Agnes Tving Stauning, Rie Louise Møller Nordestgaard, Tove Havnhøj Frandsen and Leif Percival Andersen\*

\*Address all correspondence to: leif.percival.andersen@regionh.dk

Department of Clinical Microbiology, The Helicobacter Research Center, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark

#### **References**

antibiotic resistance are in progress, but the problem is that there are many different mutations leading to the same resistance profile. *H. pylori* only develops antibiotic resistance by mutation in the genome. For MTZ, mutations in at least nine different genes are known to contribute to MTZ resistance [13]. If the detecting of MTZ resistance should be made by PCR, it would be necessary to perform the PCR with many different primers all looking for one specific mutation. In theory, this would be the most sensitive way to find MTZ resistance, but

in practice, it would be almost impossible, take a lot of time, and would be expensive.

susceptibility testing done by E-test is still the best and most economical way.

be candidates for treatment in the future [83].

102 Helicobacter Pylori - New Approaches of an Old Human Microorganism

The authors declare no conflicts of interests.

**8. Conclusion**

susceptibility testing.

**Conflict of interest**

**Author details**

and Leif Percival Andersen\*

Due to the enormous amount of mutations leading to antibiotic resistant, the culture and

The increasing resistant rates to the most commonly used antibiotics raises the question of whether other antibiotics or combinations of antibiotic and nonantibiotic should be used for primary treatment of *H. pylori* infections without susceptibility testing. Bismuth compounds in standard doses, proton pump inhibitors, and acid suppressing compounds in high doses may convert the MTZ resistance [81]. This makes MTZ useful in combination with these compounds, especially the bismuth compounds, which have been shown in clinical studies [21]. Nonantibiotics such as neuroleptics and other compounds acting on the central nerves system have anti–*H. pylori* effect *in vitro* [82] and compounds without effect on the central nervous system may be candidates for alternative treatment. Herbs like broccoli and green tee have some effect on *H. pylori* and may in combination with antibiotics and nonantibiotics

*H. pylori* is the most important gastric pathogen and may constitute the true gastric microbiota. It is, therefore, important to follow the development of resistance in *H. pylori* to antibiotics. With the increased resistance of *H. pylori* to metronidazole, clarithromycin, and levofloxacin, it may be doubtful if these antibiotics can be recommended as primary treatment without

Agnes Tving Stauning, Rie Louise Møller Nordestgaard, Tove Havnhøj Frandsen

Department of Clinical Microbiology, The Helicobacter Research Center, Copenhagen

\*Address all correspondence to: leif.percival.andersen@regionh.dk

University Hospital (Rigshospitalet), Copenhagen, Denmark


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**Chapter 8**

**Provisional chapter**

**Nonantibiotic-Based Therapeutics Targeting**

**Nonantibiotic-Based Therapeutics Targeting** 

DOI: 10.5772/intechopen.81248

The available therapy against *Helicobacter pylori* is based on a combination of antibiotics and proton pump inhibitors. The high prevalence of antibiotic-resistant strains leads to failure of this complex therapeutic regimen, leaving millions of people worldwide without effective therapeutic options. "Nature-derived" bioactive compounds with antibacterial performance may be of value for developing newer and more effective strategies. For centuries, natural compounds have played a pivotal role in traditional medicine and, in the last decades, they have gained renewed strength in the clinical field, boosted by advances in chemical characterization and extensive activity screening. Also, their recognition in gastric infection management has been empowered by the bioengineering field, namely by the development of stomach-specific delivery strategies. In this chapter, natural bioactive compounds, such as polyunsaturated fatty acids and triterpenic acids with anti-*H. pylori* effect, are described. The bioengineering approaches used to overcome

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

*Helicobacter pylori***: From Nature to the Lab**

*Helicobacter pylori***: From Nature to the Lab**

Paula Parreira, Catarina Leal Seabra,

Paula Parreira, Catarina Leal Seabra,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

their limited intrinsic bioavailability are briefly highlighted.

phytochemicals, antibiotic-free therapies

**Keywords:** nanotechnology, bioactive compounds, lipophilic compounds,

*Helicobacter pylori* is the etiologic agent of several gastric disorders that may range from chronic gastritis to more severe outcomes [1]. Ultimately, the complex interplay between *H. pylori*, the host susceptibility, and environmental factors such as smoking and drinking

Daniela Lopes-de-Campos and

http://dx.doi.org/10.5772/intechopen.81248

Maria Cristina L. Martins

**Abstract**

**1. Introduction**

Daniela Lopes-de-Campos and Maria Cristina L. Martins


#### **Chapter 8 Provisional chapter**

#### **Nonantibiotic-Based Therapeutics Targeting** *Helicobacter pylori***: From Nature to the Lab Nonantibiotic-Based Therapeutics Targeting**  *Helicobacter pylori***: From Nature to the Lab**

DOI: 10.5772/intechopen.81248

Paula Parreira, Catarina Leal Seabra, Daniela Lopes-de-Campos and Maria Cristina L. Martins Paula Parreira, Catarina Leal Seabra, Daniela Lopes-de-Campos and Maria Cristina L. Martins

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81248

#### **Abstract**

[75] Choudhari SP, Pendleton KP, Ramsey JD, et al. A systematic approach toward stabilization of CagL, a protein antigen from *Helicobacter Pylori* that is a candidate subunit vaccine.

[76] Kwok T, Zabler D, Urman S, et al. *Helicobacter* exploits integrin for type IV secretion and

[77] Chionh YT, Arulmuruganar A, Venditti E, et al. Heat shock protein complex vaccination induces protection against *Helicobacter pylori* without exogenous adjuvant. Vaccine.

[78] Nedrud JG, Bagheri N, Schön K, et al. Subcomponent vaccine based on CTA1-DD adjuvant with incorporated UreB class II peptides stimulates protective *Helicobacter pylori*

[79] D'Elios MM, Czinn SJ. Immunity, Inflammation, and Vaccines for *Helicobacter pylori*. Heli

[80] Zeng M, Mao XH, Li JX, et al. Efficacy, safety, and immunogenicity of an oral recombinant *Helicobacter pylori* vaccine in children in China: A randomised, double-blind,

[81] Chen M, Jensen B, Zhai L, et al. Nizatidine and omeprazole enhance the effect of metronidazole on *Helicobacter pylori* in vitro. International Journal of Antimicrobial Agents.

[82] Kristiansen JE, Justesen T, Hvidberg EF, et al. Trimipramine and other antipsychotics inhibit *Campylobacter pylori* in vitro. Pharmacology & Toxicology. 1989;**64**(4):386-388 [83] Fahey JW, Stephenson KK, Wallace AJ. Dietary amelioration of *Helicobacter* infection.

[84] Rahbar M, Mardanpur K, Tavafzadeh R. Imprint cytology: A simple, cost effectiveness analysis for diagnosing *Helicobacter pylori*, in west of Iran. Medical journal of the Islamic

[85] Warburton-Timms V, McNulty C. Role of screening agar plates for in vitro susceptibility testing of *Helicobacter pylori* in a routine laboratory setting. Journal of Clinical Pathology.

placebo-controlled, phase 3 trial. Lancet. 2015;**386**(10002):1457-1464

Journal of Pharmaceutical Sciences. 2013;**102**:2508-2519

immunity. Ho PL, editor. PLoS One. 2013;**8**(12):e83321

kinase activation. Nature. 2007;**449**(7164):862-866

108 Helicobacter Pylori - New Approaches of an Old Human Microorganism

2014;**32**:2350-2358

2002;**19**(3):195-200

2001;**54**(5):408-411

cobacter. 2014;**19**(S1):19-261

Nutrition Research. 2015;**35**(6):461-473

Republic of Iran. 2012;**26**(1):12-16

The available therapy against *Helicobacter pylori* is based on a combination of antibiotics and proton pump inhibitors. The high prevalence of antibiotic-resistant strains leads to failure of this complex therapeutic regimen, leaving millions of people worldwide without effective therapeutic options. "Nature-derived" bioactive compounds with antibacterial performance may be of value for developing newer and more effective strategies. For centuries, natural compounds have played a pivotal role in traditional medicine and, in the last decades, they have gained renewed strength in the clinical field, boosted by advances in chemical characterization and extensive activity screening. Also, their recognition in gastric infection management has been empowered by the bioengineering field, namely by the development of stomach-specific delivery strategies. In this chapter, natural bioactive compounds, such as polyunsaturated fatty acids and triterpenic acids with anti-*H. pylori* effect, are described. The bioengineering approaches used to overcome their limited intrinsic bioavailability are briefly highlighted.

**Keywords:** nanotechnology, bioactive compounds, lipophilic compounds, phytochemicals, antibiotic-free therapies

#### **1. Introduction**

*Helicobacter pylori* is the etiologic agent of several gastric disorders that may range from chronic gastritis to more severe outcomes [1]. Ultimately, the complex interplay between *H. pylori*, the host susceptibility, and environmental factors such as smoking and drinking

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

can lead to gastric cancer, which is the fifth most common cancer worldwide, accounting for 754,000 deaths in 2015 [2, 3]. Despite significant medical advances, the 5-year survival rate from gastric cancer is low (31%), mainly because this cancer is diagnosed at later stages [4]. It is widely recognized that the best strategy to reduce the risk of gastric carcinoma associated with *H. pylori* infection is its eradication from infected hosts [5, 6]. The current treatment relies on a combination of antibiotics (clarithromycin plus amoxicillin or metronidazole) and an acid-suppressive drug (e.g., proton-pump inhibitor), since no available substances are effective as monotherapy [7]. However, the eradication rates of this therapeutic scheme have been declining to unacceptable levels [8], mostly due to high antibiotic resistance levels. In fact, *H. pylori* has been placed among the 16 antibiotic-resistant bacteria that pose greatest threat to human health [9]. It is noteworthy that besides resistance and coinfection with multiple strains with distinct antibiotic susceptibilities, other factors also account for conventional treatment failure:


In this scenario, where *H. pylori* traditional antibiotic therapies fail, a considerable interest in alternative therapeutic players combined with bioengineering strategies has arisen. Several "nature-derived" options have been studied [5], and some of them are summarized in **Figure 1**.

Although very promising *in vitro*, these molecules share a common drawback when transferred to *in vivo* settings: intrinsic limited bioavailability. **Figure 2** summarizes some of the bioengineering approaches envisioned to overcome the mentioned limitation.

Chitosan micro-/nanoparticles have been extensively studied as drug delivery systems targeting *H. pylori* infection [16], mainly due to its gastric retentive properties [17]. Chitosan, a natural biocompatible polysaccharide obtained by *N*-deacetylation of chitin [18], has mucoadhesive properties due to electrostatic interactions between its cationic amine groups and gastric mucins, which are negatively charged at the acidic gastric pH [19, 20].

action [21]. Polyelectrolyte complex nanoparticles (PNPs), prepared by the combination of chitosan with negatively charged polymers, alginate or heparin, have also been used to encapsulate the antimicrobial peptide pexiganan [22] and berberine [23], respectively. This approach increased the effectiveness of these bioactive compounds, inhibiting *H. pylori* growth

**Figure 2.** Most common bioengineering approaches applicable to "nature-derived" bioactives in the scope of *H. pylori*

**Figure 1.** Some "nature-derived" bioactive compounds described in the literature in the scope of novel anti*-H. pylori*

Nonantibiotic-Based Therapeutics Targeting *Helicobacter pylori*: From Nature to the Lab

http://dx.doi.org/10.5772/intechopen.81248

and reducing their cytotoxic effects.

infection management.

therapeutics.

Chitosan microspheres were used to encapsulate and improve the biological activity of transresveratrol, a phenolic compound that has, among other biological activities, anti-*H. pylori* Nonantibiotic-Based Therapeutics Targeting *Helicobacter pylori*: From Nature to the Lab http://dx.doi.org/10.5772/intechopen.81248 111

can lead to gastric cancer, which is the fifth most common cancer worldwide, accounting for 754,000 deaths in 2015 [2, 3]. Despite significant medical advances, the 5-year survival rate from gastric cancer is low (31%), mainly because this cancer is diagnosed at later stages [4]. It is widely recognized that the best strategy to reduce the risk of gastric carcinoma associated with *H. pylori* infection is its eradication from infected hosts [5, 6]. The current treatment relies on a combination of antibiotics (clarithromycin plus amoxicillin or metronidazole) and an acid-suppressive drug (e.g., proton-pump inhibitor), since no available substances are effective as monotherapy [7]. However, the eradication rates of this therapeutic scheme have been declining to unacceptable levels [8], mostly due to high antibiotic resistance levels. In fact, *H. pylori* has been placed among the 16 antibiotic-resistant bacteria that pose greatest threat to human health [9]. It is noteworthy that besides resistance and coinfection with multiple strains with distinct antibiotic susceptibilities, other factors also account for conventional

**a.** drugs bioavailability: antibiotics are typically administrated via the oral route. Since the gastric mucus layer acts as a barrier to antibiotic delivery, most drugs are unable to reach

**b.** gastric features: the pH at the stomach/duodenum varies from acidic to neutral, depending on the presence or absence of food and the location within the mucus barrier that covers the epithelial cells. This affects the efficacy of most antibiotics once only a few remain

**c.** compliance: side effects, such as taste disturbances with clarithromycin and metronidazole, and diarrhea with amoxicillin, account for the poor patient compliance. Additionally, complex regimens that require multiple doses of medication each day also decrease thera-

**d.** lifestyle: smoking and alcohol consumption are thought to contribute for treatment

In this scenario, where *H. pylori* traditional antibiotic therapies fail, a considerable interest in alternative therapeutic players combined with bioengineering strategies has arisen. Several "nature-derived" options have been studied [5], and some of them are summarized

Although very promising *in vitro*, these molecules share a common drawback when transferred to *in vivo* settings: intrinsic limited bioavailability. **Figure 2** summarizes some of the

Chitosan micro-/nanoparticles have been extensively studied as drug delivery systems targeting *H. pylori* infection [16], mainly due to its gastric retentive properties [17]. Chitosan, a natural biocompatible polysaccharide obtained by *N*-deacetylation of chitin [18], has mucoadhesive properties due to electrostatic interactions between its cationic amine groups and

Chitosan microspheres were used to encapsulate and improve the biological activity of transresveratrol, a phenolic compound that has, among other biological activities, anti-*H. pylori*

bioengineering approaches envisioned to overcome the mentioned limitation.

gastric mucins, which are negatively charged at the acidic gastric pH [19, 20].

the underlying gastric epithelium, where *H. pylori* is attached [10];

treatment failure:

active in a wide pH range [11–13];

110 Helicobacter Pylori - New Approaches of an Old Human Microorganism

peutic compliance [14];

failure [15].

in **Figure 1**.

**Figure 1.** Some "nature-derived" bioactive compounds described in the literature in the scope of novel anti*-H. pylori* therapeutics.

**Figure 2.** Most common bioengineering approaches applicable to "nature-derived" bioactives in the scope of *H. pylori* infection management.

action [21]. Polyelectrolyte complex nanoparticles (PNPs), prepared by the combination of chitosan with negatively charged polymers, alginate or heparin, have also been used to encapsulate the antimicrobial peptide pexiganan [22] and berberine [23], respectively. This approach increased the effectiveness of these bioactive compounds, inhibiting *H. pylori* growth and reducing their cytotoxic effects.

Other strategies that can be used to overcome the low bioavailability and solubility of lipophilic compounds comprise ionic liquids (IL) and lipid nanoparticles.

membrane as the main target of fatty acids, leading to a sequence of biophysical phenomena including membrane destabilization, pore formation, and lysis of bacteria [32, 41, 44, 45]. The multiple mechanisms that are behind their ability to perturb bacterial cell membranes lead to a low probability of antimicrobial resistance [34]. On the opposite, small molecules, such as commercial antibiotics, inhibit specific enzymes and, consequently, increase the probability of

Nonantibiotic-Based Therapeutics Targeting *Helicobacter pylori*: From Nature to the Lab

http://dx.doi.org/10.5772/intechopen.81248

The antibacterial activity of PUFAs depends on their molecular structure. In fact, the existence of double bonds and, more specifically, their number and orientation within the fatty acids are important for their physicochemical properties [46, 47]. These structural differences are reported to affect their ability to inhibit *H. pylori* growth *in vitro* [45]. For instance, the inhibitory effect is higher for higher degrees of unsaturation: [oleic (C18:1) < linoleic (C18:2) < arachidonic (C20:4) < n-3 linolenic (C18:3) = n-6 linolenic (C18:3) = eicosapentaenoic (C20:5) acid] [45]. The encapsulation of free fatty acids in nanoparticles can improve their pharmacokinetic and pharmacological properties [34]. A review of the nanotechnology formulations used to

In this section, two of the most promising fatty acids (docosahexaenoic acid (DHA) and lino-

**Figure 3** illustrates the two bioengineering approaches that have been applied to DHA and

DHA inhibits *H. pylori* growth both *in vitro* and *in vivo*, since it is able to reduce *H pylori* adhesion to gastric cells and bacterial ATP production [42, 43, 48]. DHA induces changes in expression of *H. pylori* outer membrane proteins associated with stress response, metabolism, and modified bacterial lipopolysaccharide phenotype [43]. DHA is also able to indirectly interfere with *H. pylori* growth since it alters cholesterol levels in epithelial cells, thereby influencing

lenic acid (LA)) are described regarding their specific application against *H. pylori*.

antimicrobial resistance development [34].

encapsulate antimicrobial lipids was recently published [34].

LA that will be discussed in the following subsections.

the bacterium ability to uptake and use epithelial cholesterol [48].

**Figure 3.** Encapsulation of fatty acids, namely DHA and LA, in different types of nanoparticles.

**2.1. Docosahexaenoic acid (DHA)**

IL are a new class of powerful catanionic hydrotropes, where both the cation and the anion synergistically contribute to increase the solubility of biomolecules in water [24]. Therefore, IL enhance the solubility of hydrophobic substances in aqueous media and are widely used in the formulation of drugs, cleaning, and personal care products. IL have been explored to increase the water solubility of triterpenic acids, such as of betulinic acid [25, 26].

Lipid nanoparticles are very useful to encapsulate lipophilic compounds due to their higher biocompatibility compared to polymeric nanoparticles [27, 28]. Liposomes, firstly described in the mid-1960s, are sphere-shaped vesicles consisting of one or more phospholipid bilayers. Liposomes are the first nanodrug delivery systems that have been successfully translated into real-time clinical application [29–31]. Nanostructured lipid carriers (NLCs) are lipid nanoparticles specifically designed and patented as drug delivery systems, and they are characterized by a solid-lipid core composed of a mixture of solid and liquid lipids [32]. NLCs can be prepared using a wide variety of lipids including fatty acids, glyceride mixtures, or waxes, stabilized with biocompatible surfactants, which makes this a very versatile strategy. Both are considered safe and under the Generally Recognized as Safe (GRAS) status issued by the Food and Drug Administration (FDA) [31, 33].

Polyunsaturated fatty acids and triterpenic acids with anti-*H. pylori* effect have gained renewed interest in the scientific community as alternatives to overcome the increasing number of drug-resistant bacteria. These lipophilic bioactive compounds can largely benefit from a nanotechnological approach to improve their stability and to overcome their limited intrinsic bioavailability and thus, they will be briefly highlighted in the next sections.

#### **2. "Nature-derived" anti-***H. pylori* **fatty acids**

Free fatty acids, also known as antimicrobial lipids [34], are linear carbon chains, which are the main constituent of phospholipids, triglycerides, sterol esters, among others [35]. Consequently, they are important for biological activities, such as for energetic, metabolic, and structural processes [35]. Fatty acids are classified according to the length of the carbon chains, the number of double bonds, and their positions within the moiety [35]. Polyunsaturated fatty acids (PUFAs) have two or more double bonds [36], and they have been recognized for their broad-spectrum activity against bacteria (e.g., *H. pylori*), fungi, protozoa, and virus [36–38]. This is due to the ability of fatty acids to work as mild surfactants [34]. The disturbance of the bacterial cell membrane can lead to the deregulation of metabolic pathways, inhibiting the bacterial growth, or even to lysis and death [34]. The specific interaction between antimicrobial fatty acids and bacterial membranes remains to be fully understood. Some studies revealed that free fatty acids can induce different kinds of morphological changes in the membrane [39, 40]. Khulusi et al. and Correia et al. reported that fatty acids can be incorporated into *H. pylori* phospholipids membrane, being able to change the bacillary morphology of the bacteria to their coccoid shape [32, 41–43]. Several studies also identified the bacterial membrane as the main target of fatty acids, leading to a sequence of biophysical phenomena including membrane destabilization, pore formation, and lysis of bacteria [32, 41, 44, 45]. The multiple mechanisms that are behind their ability to perturb bacterial cell membranes lead to a low probability of antimicrobial resistance [34]. On the opposite, small molecules, such as commercial antibiotics, inhibit specific enzymes and, consequently, increase the probability of antimicrobial resistance development [34].

The antibacterial activity of PUFAs depends on their molecular structure. In fact, the existence of double bonds and, more specifically, their number and orientation within the fatty acids are important for their physicochemical properties [46, 47]. These structural differences are reported to affect their ability to inhibit *H. pylori* growth *in vitro* [45]. For instance, the inhibitory effect is higher for higher degrees of unsaturation: [oleic (C18:1) < linoleic (C18:2) < arachidonic (C20:4) < n-3 linolenic (C18:3) = n-6 linolenic (C18:3) = eicosapentaenoic (C20:5) acid] [45]. The encapsulation of free fatty acids in nanoparticles can improve their pharmacokinetic and pharmacological properties [34]. A review of the nanotechnology formulations used to encapsulate antimicrobial lipids was recently published [34].

In this section, two of the most promising fatty acids (docosahexaenoic acid (DHA) and linolenic acid (LA)) are described regarding their specific application against *H. pylori*.

**Figure 3** illustrates the two bioengineering approaches that have been applied to DHA and LA that will be discussed in the following subsections.

#### **2.1. Docosahexaenoic acid (DHA)**

Other strategies that can be used to overcome the low bioavailability and solubility of lipo-

IL are a new class of powerful catanionic hydrotropes, where both the cation and the anion synergistically contribute to increase the solubility of biomolecules in water [24]. Therefore, IL enhance the solubility of hydrophobic substances in aqueous media and are widely used in the formulation of drugs, cleaning, and personal care products. IL have been explored to

Lipid nanoparticles are very useful to encapsulate lipophilic compounds due to their higher biocompatibility compared to polymeric nanoparticles [27, 28]. Liposomes, firstly described in the mid-1960s, are sphere-shaped vesicles consisting of one or more phospholipid bilayers. Liposomes are the first nanodrug delivery systems that have been successfully translated into real-time clinical application [29–31]. Nanostructured lipid carriers (NLCs) are lipid nanoparticles specifically designed and patented as drug delivery systems, and they are characterized by a solid-lipid core composed of a mixture of solid and liquid lipids [32]. NLCs can be prepared using a wide variety of lipids including fatty acids, glyceride mixtures, or waxes, stabilized with biocompatible surfactants, which makes this a very versatile strategy. Both are considered safe and under the Generally Recognized as Safe (GRAS) status issued by the

Polyunsaturated fatty acids and triterpenic acids with anti-*H. pylori* effect have gained renewed interest in the scientific community as alternatives to overcome the increasing number of drug-resistant bacteria. These lipophilic bioactive compounds can largely benefit from a nanotechnological approach to improve their stability and to overcome their limited intrin-

Free fatty acids, also known as antimicrobial lipids [34], are linear carbon chains, which are the main constituent of phospholipids, triglycerides, sterol esters, among others [35]. Consequently, they are important for biological activities, such as for energetic, metabolic, and structural processes [35]. Fatty acids are classified according to the length of the carbon chains, the number of double bonds, and their positions within the moiety [35]. Polyunsaturated fatty acids (PUFAs) have two or more double bonds [36], and they have been recognized for their broad-spectrum activity against bacteria (e.g., *H. pylori*), fungi, protozoa, and virus [36–38]. This is due to the ability of fatty acids to work as mild surfactants [34]. The disturbance of the bacterial cell membrane can lead to the deregulation of metabolic pathways, inhibiting the bacterial growth, or even to lysis and death [34]. The specific interaction between antimicrobial fatty acids and bacterial membranes remains to be fully understood. Some studies revealed that free fatty acids can induce different kinds of morphological changes in the membrane [39, 40]. Khulusi et al. and Correia et al. reported that fatty acids can be incorporated into *H. pylori* phospholipids membrane, being able to change the bacillary morphology of the bacteria to their coccoid shape [32, 41–43]. Several studies also identified the bacterial

sic bioavailability and thus, they will be briefly highlighted in the next sections.

increase the water solubility of triterpenic acids, such as of betulinic acid [25, 26].

philic compounds comprise ionic liquids (IL) and lipid nanoparticles.

112 Helicobacter Pylori - New Approaches of an Old Human Microorganism

Food and Drug Administration (FDA) [31, 33].

**2. "Nature-derived" anti-***H. pylori* **fatty acids**

DHA inhibits *H. pylori* growth both *in vitro* and *in vivo*, since it is able to reduce *H pylori* adhesion to gastric cells and bacterial ATP production [42, 43, 48]. DHA induces changes in expression of *H. pylori* outer membrane proteins associated with stress response, metabolism, and modified bacterial lipopolysaccharide phenotype [43]. DHA is also able to indirectly interfere with *H. pylori* growth since it alters cholesterol levels in epithelial cells, thereby influencing the bacterium ability to uptake and use epithelial cholesterol [48].

**Figure 3.** Encapsulation of fatty acids, namely DHA and LA, in different types of nanoparticles.

Although *in vivo* studies using gastric-infected mice demonstrated that DHA was able to decrease only 50% of *H. pylori* gastric colonization, the DHA conjugation with antibiotic standard treatment decreased the recurrence of *H. pylori* infection [42, 43].

bacterial membrane, being directly and faster incorporated into the bacterial membrane [57]. Their main mechanism is the increase of the permeability of the outer membrane and of the plasma membrane of *H. pylori,* which leads to a leakage of cytoplasmic contents [58]. They also decrease the *H. pylori-*induced proinflammatory cytokines, helping in the healing of the gastric mucosa [58]. Furthermore, the ability of those liposomes to be retained at the site of infection was also shown *in vivo* [53]. The retention for up to 24 h was attributed to the small size of the liposomes and their anionic surface charge, which decreases their hydrophobic entrapment [53]. The biocompatibility of the formulation was shown through gastric histopathology and mucosal integrity and by the maintenance of the gut microbiota, on the opposite

Nonantibiotic-Based Therapeutics Targeting *Helicobacter pylori*: From Nature to the Lab

http://dx.doi.org/10.5772/intechopen.81248

Phytochemicals have been used for centuries in the treatment of gastrointestinal disorders, such as dyspepsia, gastritis, and peptic ulcer disease [60]. Over the last two decades, phytotherapy has gained strength in the scientific community, prompted by the need of alternatives

Plants synthesize a vast range of secondary metabolites with a significant portion consisting of phenolic and flavonoid compounds [61]. These secondary metabolites, other than providing plants with unique survival or adaptive strategies, are associated to a wide range of biological activities [62]. Phenolic compounds, namely wine polyphenols, from which resveratrol is the most studied, and olive oil polyphenols, mainly hydroxytyrosol, have been associated with anti-*H. pylori* activity [5]. Lipophilic compounds from the terpenes family can also be obtained from several plants. In the scope of anti-*H. pylori* strategies, these are described in more detail

of isoprene building blocks, they are classified into several groups, such as monoterpenes, sesquiterpenes, diterpenes, triterpenes, and tetraterpenes (with 2, 3, 4, 6, and 8 isoprene units, respectively). These compounds can undergo chemical modifications by oxidation or rearrangement of the carbon skeleton, which leads to a vast group of compounds denominated

Pentacyclic triterpenoids are commonly isolated as active substances from different natural sources, mainly plant surfaces such as stem bark or leaf and fruit waxes [64]. Among them, pentacyclic triterpenes (C30H48) are being marketed as therapeutic agents or dietary supplements around the world due to their biological applications [65, 66]. Their antibacterial properties are also recognized. For instance, it was demonstrated that the acidic fraction of the total mastic extract without polymer (TMEWP) from the Chios Mastic Gum (resin of

)n), where n is the number of isoprene units. Depending on the number

H8

)n (▬(▬CH2

Terpenes are naturally occurring hydrocarbons, with the general formula (C5

of the current triple therapy [53, 59].

to the ineffectiveness of traditional antibiotics.

in the following section.

)▬CH〓CH2

**3.1. Triterpenic acids**

〓C(CH3

terpenoids [63].

**3. "Nature-derived" anti-***H. pylori* **phytochemicals**

Another DHA feature is its ability to attenuate the host inflammatory response associated with gastric infection [49, 50].

DHA poor solubility in water, fast oxidation/degradation plus gastric settings drawbacks (namely low gastric residence time and low penetration through the gastric mucus layer) are challenging issues for its clinical translation [36, 50, 51]. To overcome these obstacles, cytocompatible lipid nanoparticles have been researched to encapsulate DHA [44, 52]. It was demonstrated that DHA lipid nanoparticles are able to destabilize *H. pylori* membranes, leading to disruption and leakage of cytoplasmic contents [32, 44]. Importantly, these lipid nanoparticles do not interfere with normal gut microbiota in opposite to dramatic changes described for the conventional antibiotic therapy [44].

#### **2.2. Linolenic acid (LA)**

LA, as fatty acids in general, is considered safe [53]. It is classified as an essential fatty acid, once it cannot be synthesized by the human body, being necessary to be supplied by the diet [54]. Its importance for biological processes is unquestionable. LA undergoes metabolic changes *in vivo* that ultimately lead to the formation of prostaglandins, thromboxanes, leukotrienes, and lipoxins [54]. Furthermore, the usefulness of LA as an antibacterial agent was also proved, being one of the most potent unsaturated fatty acids against *H. pylori* [7]. It also promotes the adhesion of *Lactobacillus casei* to mucosa surfaces, which indirectly hinders the growth of *H. pylori* [55]. Besides its bactericidal effect, LA is also important for the integrity of the gastric mucosa. It was already proposed that lower levels of essential fatty acids, such as LA, lead to decreased levels of prostaglandins and, consequently, to a higher susceptibility of the gastric mucosa to ulcerogenic agents [56].

Nanotechnology has been successfully used to load fatty acids, including LA [34]. As above mentioned, the oral administration of fatty acids is hindered by their poor solubility, especially at acidic pH, and their susceptibility to chemical degradation [57]. In fact, the carboxyl protonation under acidic pH at the stomach lumen decreases the efficacy of fatty acids after oral administration [53]. This was already shown *in vivo*, with no significant effect of plain LA in killing *H. pylori* on a mouse model [53]. Nevertheless, liposomes are promising bioengineering strategies to overcome these limitations. Due to the amphiphilic nature of fatty acids, they can be easily incorporated into the phospholipid bilayer of liposomes [57]. Hence, Obonyo et al. used liposomes of egg phosphatidylcholines, cholesterol, and LA to kill *H. pylori* [57]. They showed that LA-loaded liposomes were effective against *H. pylori* even in its coccoid form and regardless their resistance to antibiotics [57]. Interestingly, *H. pylori* developed resistance against free LA at subbactericidal concentrations, whereas it showed no resistance against LA when incorporated into the nanoparticles [57]. These results show the promising usefulness of nanotechnology not only to protect the fatty acid from its degradation, but also to improve its efficacy. The higher efficacy relies on their ability to fuse with the bacterial membrane, being directly and faster incorporated into the bacterial membrane [57]. Their main mechanism is the increase of the permeability of the outer membrane and of the plasma membrane of *H. pylori,* which leads to a leakage of cytoplasmic contents [58]. They also decrease the *H. pylori-*induced proinflammatory cytokines, helping in the healing of the gastric mucosa [58]. Furthermore, the ability of those liposomes to be retained at the site of infection was also shown *in vivo* [53]. The retention for up to 24 h was attributed to the small size of the liposomes and their anionic surface charge, which decreases their hydrophobic entrapment [53]. The biocompatibility of the formulation was shown through gastric histopathology and mucosal integrity and by the maintenance of the gut microbiota, on the opposite of the current triple therapy [53, 59].
