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ABSTRACT
The human peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. It has been shown to protect against gastritis caused by Helicobacter pylori infection. Most of the current synthetic expression systems for LL-37 depend on the construction of soluble fusion partners to avoid cytotoxic effects of the antimicrobial peptide in the E. coli host strain. However, the fusion systems require additional cleavage steps using enzymatic or chemical methods, which makes them impossible to express an active LL-37 peptide in vivo. In order to create a resistant host that can export LL-37 to the media, we intend to overexpress the E. coli marRAB operon, which activates the AcrAB-TolC efflux pump, a mechanism that has been related with resistance to this and similar antimicrobial peptides by expulsion. Our aim is to create a system in which E. coli expels LL-37 only when H. pylori and other pathogenic bacteria are present. In order to do this, we are using the LsrA promoter, which allows transcription in presence of AI-2, a molecule produced by bacteria to communicate via quorum-sensing.
CONTENTS
1. The Problem
1.1 Etiologic Agent
1.2 Epidemiology
1.3 Pathology and Pathogenesis
1.4 Diagnosis and prescription
1.5 Treatment: H. Pylori Infections
1.6 Resistance
2. The Solution
2.1 Using quorum sensing to regulate gene expression
2.2 Antimicrobial Peptide
2.3 Resistance
THE PROBLEM
Helicobacter pylori colonizes the stomach of 50% of the world's human population throughout their lifetimes. Colonization with this organism is the main risk factor for peptic ulceration as well as for gastric adenocarcinoma and gastric MALT (mucosa-associated lymphoid tissue) lymphoma. Treatment for H. pylori has revolutionized the management of peptic ulcer disease, providing a permanent cure in most cases. Such treatment also represents first-line therapy for patients with low-grade gastric MALT lymphoma. Treatment of H. pylori is of no benefit in the treatment of gastric adenocarcinoma, but prevention of H. pylori colonization could potentially prevent gastric malignancy and peptic ulceration [1,2]
Etiologic Agent
H. pylori is a gram-negative bacillus that has naturally colonized humans for at least 50,000 years—and probably throughout human evolution. It lives in gastric mucus, with a small proportion of the bacteria adherent to the mucosa and possibly a very small number of the organisms entering cells or penetrating the mucosa; its distribution is never systemic. Its spiral shape and flagella render H. pylori motile in the mucus environment. The organism has several acid-resistance mechanisms, most notably a highly expressed urease that catalyzes urea hydrolysis to produce buffering ammonia. H. pylori is microaerophilic (requiring low levels of oxygen), is slow-growing, and requires complex growth media in vitro. Publication of several complete genomic sequences of H. pylori since 1997 has led to significant advances in the understanding of the organism's biology [3].
A very small proportion of gastric Helicobacter infections are due to species other than H. pylori, possibly acquired as zoonoses. Whether these non-pylori gastric helicobacters cause disease remains controversial. In immunocompromised hosts, several nongastric (intestinal) Helicobacter species can cause disease with clinical features resembling those of Campylobacter infections.
Epidemiology
The prevalence of H. pylori among adults is 30% in the United States and other developed countries as opposed to >80% in most developing countries. In the United States, prevalence varies with age: 50% of 60-year-old persons, 20% of 30-year-old persons, and <10% of children are colonized.H. pylori is usually acquired in childhood. The age association is due mostly to a birth-cohort effect whereby current 60-year-olds were more commonly colonized as children than are current children. Spontaneous acquisition or loss of H. pylori in adulthood is uncommon. Other strong risk factors for H. pylori colonization are markers of crowding and maternal colonization. The low incidence among children in developed countries at present is due, at least in part, to decreased maternal colonization and increased use of antibiotics.
Humans are the only important reservoir of H. pylori. Children may acquire the organism from their parents (more often from the mother) or from other children. Whether transmission takes place more often by the fecal-oral or the oral-oral route is unknown, but H. pylori is easily cultured from vomitus and gastroesophageal refluxate and is less easily cultured from stool.
In Mexico, seroprevalence studies were positive in 20% of children under 1 year, 50% at 10 years and 80% in those over 25 years [11, 12].
Pathology and Pathogenesis
H. pylori colonization induces a tissue response in the stomach, chronic superficial gastritis, which includes infiltration of the mucosa by both mononuclear and polymorphonuclear cells. (The term gastritis should be used specifically to describe histologic features; it has also been used to describe endoscopic appearances and even symptoms, which do not correlate with microscopic findings or even with the presence of H. pylori.) Although H. pylori is capable of numerous adaptations that prevent excessive stimulation of the immune system, colonization is accompanied by a considerable persistent immune response, including the production of both local and systemic antibodies as well as cell-mediated responses. However, these responses are ineffective in clearing the bacterium. This inefficient clearing appears to be due in part to H. pylori's downregulation of the immune system, which fosters its own persistence.
The pattern of gastric inflammation is associated with disease risk: antral-predominant gastritis is most closely linked with duodenal ulceration, whereas pangastritis is linked with gastric ulceration and adenocarcinoma. This difference probably explains why patients with duodenal ulceration are not at high risk of developing gastric adenocarcinoma later in life, despite being colonized by H. pylori.
How gastric colonization causes duodenal ulceration is now becoming clearer. H. pylori–induced inflammation diminishes the number of somatostatin-producing D cells. Since somatostatin inhibits gastrin release, gastrin levels are higher than in H. pylori–negative persons, and these higher levels lead to increased meal-stimulated acid secretion in the gastric corpus, which is only mildly inflamed in antral-predominant gastritis. How this increases duodenal ulcer risk remains controversial, but the increased acid secretion may contribute to the formation of the potentially protective gastric metaplasia found in the duodenum of duodenal ulcer patients. Gastric metaplasia in the duodenum may become colonized by H. pylori and subsequently inflamed and ulcerated.
The pathogenesis of gastric ulceration and that of gastric adenocarcinoma are less well understood, although both conditions arise in association with pan- or corpus-predominant gastritis. The hormonal changes described above still occur, but the inflammation in the gastric corpus means that it produces less acid (hypochlorhydria) despite hypergastrinemia. Gastric ulcers usually occur at the junction of antral and corpus-type mucosa, and this region is particularly inflamed. Gastric cancer probably stems from progressive DNA damage and the survival of abnormal epithelial cell clones. The DNA damage is thought to be due principally to reactive oxygen and nitrogen species arising from inflammatory cells and perhaps in relation to other bacteria that survive in a hypochlorhydric stomach. Longitudinal analyses of gastric biopsy specimens taken years apart from the same patient show that the common intestinal type of gastric adenocarcinoma follows stepwise changes from simple gastritis to gastric atrophy, intestinal metaplasia, and dysplasia. A second, diffuse type of gastric adenocarcinoma may arise directly from chronic gastritis alone.
Diagnosis and prescription
Tests for the presence of H. pylori can be divided into two groups: invasive tests, which require upper gastrointestinal endoscopy and are based on the analysis of gastric biopsy specimens, and noninvasive tests. Endoscopy often is not performed in the initial management of young dyspeptic patients without "alarm" symptoms but is commonly used to exclude malignancy in older patients. If endoscopy is performed, the most convenient biopsy-based test is the biopsy urease test, in which one large or two small antral biopsy specimens are placed into a gel containing urea and an indicator. The presence of H. pylori urease leads to a pH alteration and therefore to a color change, which often occurs within minutes but can require up to 24 h. Histologic examination of biopsy specimens for H. pylori also is accurate, provided that a special stain (e.g., a modified Giemsa or silver stain) permitting optimal visualization of the organism is used. If biopsy specimens are obtained from both antrum and corpus, histologic study yields additional information, including the degree and pattern of inflammation, atrophy, metaplasia, and dysplasia. Microbiologic culture is most specific but may be insensitive because of difficulty with H. pylori isolation. Once the organism is cultured, its identity as H. pylori can be confirmed by its typical appearance on Gram's stain and its positive reactions in oxidase, catalase, and urease tests. Moreover, the organism's susceptibility to antibiotics can be determined, and this information can be clinically useful in difficult cases. The occasional biopsy specimens containing the less common non-pylori gastric helicobacters give only weakly positive results in the biopsy urease test. Positive identification of these bacteria requires visualization of the characteristic long, tight spirals in histologic sections [4,5].
| Tests Commonly Used to Detect Helicobacter pylori | ||
|---|---|---|
| Test | Advantages | Disadvantages |
| Invasive (Based on Endoscopic Biopsy) | ||
| Biopsy urease test | Quick, simple | Some commercial tests not fully sensitive before 24 h |
| Histology | May give additional histologic information | Sensitivity dependent on experience and use of special stains |
| Culture | Permits determination of antibiotic susceptibility | Sensitivity dependent on experience |
| Noninvasive | ||
| Serology | Inexpensive and convenient; not affected by recent antibiotics or proton pump inhibitors to the same extent as breath and stool tests | Cannot be used for early follow-up after treatment; some commercial kits inaccurate, and all less accurate than breath test |
| 13C urea breath test | Inexpensive and simpler than endoscopy; useful for follow-up after treatment | Requires fasting; not as convenient as blood or stool tests |
| Stool antigen test | Inexpensive and convenient; useful for follow-up after treatment; may be useful in children | May be disliked by people from some cultures; may be slightly less accurate than urea breath test, particularly when used to assess treatment success |
Noninvasive H. pylori testing is the norm if gastric cancer does not need to be excluded by endoscopy. The most consistently accurate test is the urea breath test. In this simple test, the patient drinks a solution of urea labeled with the nonradioactive isotope 13C and then blows into a tube. If H. pylori urease is present, the urea is hydrolyzed and labeled carbon dioxide is detected in breath samples. The stool antigen test, another simple assay, is more convenient and potentially less expensive than the urea breath test but has been slightly less accurate in some comparative studies. The simplest tests for ascertaining H. pylori status are serologic assays measuring specific IgG levels in serum by enzyme-linked immunosorbent assay or immunoblot. The best of these tests are as accurate as other diagnostic methods, but many commercial tests—especially rapid office tests—do not perform well.
Treatment: H. pylori Infections
The most clear-cut indications for treatment are H. pylori–related duodenal or gastric ulceration or low-grade gastric B cell lymphoma. H. pylori should be eradicated in patients with documented ulcer disease, whether or not the ulcers are currently active, to reduce the likelihood of relapse. Many guidelines now recommend H. pylori eradication in uninvestigated simple dyspepsia following noninvasive diagnosis; others also recommend treatment in functional dyspepsia, in case the patient is one of the perhaps 7% (beyond placebo effects) to benefit from such treatment. Individuals with a strong family history of gastric cancer should be treated to eradicate H. pylori in the hope that their cancer risk will be reduced. Currently, widespread community screening for and treatment of H. pylori as primary prophylaxis for gastric cancer and peptic ulcers are not recommended, mainly because it is unclear whether treatment for H. pylori reduces the risk of cancer to that in persons who have never acquired the organism. The largest randomized controlled study to date (performed in China) showed no cancer risk reduction during the 7 years of follow-up, although a post hoc subgroup analysis documented improvement in the group of participants who did not already have gastric atrophy or intestinal metaplasia. Other studies have found a reduced cancer risk after treatment, but the size of this effect in different populations remains unclear, and the results of further large-scale prospective interventional studies must be awaited. Other reasons for not treating H. pylori in asymptomatic populations at present include:
- The adverse side effects of the multiple-antibiotic regimens used. (which are common and can be severe in rare cases)
- Antibiotic resistance, which may arise in H. pylori or other incidentally carried bacteria
- The anxiety that may arise in otherwise healthy people, especially if treatment is unsuccessful.
- The apparent existence of a subset of people who will develop GERD symptoms after treatment, although on average H. pylori treatment does not affect GERD symptoms or severity.
Although H. pylori is susceptible to a wide range of antibiotics in vitro, monotherapy is not usually successful, probably because of inadequate antibiotic delivery to the colonization niche. Failure of monotherapy has prompted the development of multidrug regimens, the most successful of which are triple and quadruple combinations. Initially these regimens produced H. pylori eradication rates of >90% in many trials; in recent years, however, resistance to key antibiotics has become more common, a trend leading to H. pylori eradication rates of only 75–80% for the most commonly used regimens. Current regimens consist of a PPI or ranitidine bismuth citrate and two or three antimicrobial agents given for 7–14 days. Research on optimizing drug combinations to increase efficacy continues, and it is likely that guidelines will change as the field develops and as countries increasingly individualize treatment to suit local antibiotic resistance patterns and economic needs [6,7].
An increasing number of infected individuals are found to harbor antibiotic-resistant bacteria. This results in initial treatment failure and requires additional rounds of antibiotic therapy or alternative strategies, such as a quadruple therapy.[8,9]
| Recommended Treatment Regimens for Helicobacter pylori | ||||
|---|---|---|---|---|
| Regimen (Duration) | Drug 1 | Drug 2 | Drug 3 | Drug 4 |
| Regimen 1: OCM (7–14 days)a | Omeprazoleb (20 mg bid) | Clarithromycin (500 mg bid) | Metronidazole (500 mg bid) | — |
| Regimen 2: OCA (7–14 days)a | Omeprazoleb (20 mg bid) | Clarithromycin (500 mg bid) | Amoxicillin (1 g bid) | — |
| Regimen 3: OBTM (14 days)c | Omeprazoleb (20 mg bid) | Bismuth subsalicylate (2 tabs qid) | Tetracycline HCl (500 mg qid) | Metronidazole (500 mg tid) |
| Regimen 4d: sequential (5 days + 5 days) | Omeprazoleb (20 mg bid) | Amoxicillin 1 g bid | — | — |
| Omeprazoleb (20 mg bid) | Clarithromycin (500 mg bid) | Tinidazole (500 mg bid) | — | |
| Regimen 5e: OAL (10 days) | Omeprazoleb (20 mg bid) | Amoxicillin (1 g bid) | Levofloxacin (500 mg qid) | — |
- a Meta-analyses show that a 14-day course of therapy is slightly superior to a 7-day course. However, in populations where 7-day treatment is known to have very high success rates, this shorter course is still often used.
- b Omeprazole may be replaced with any proton pump inhibitor at an equivalent dosage or, in regimens 1 and 2, with ranitidine bismuth citrate (400 mg).
- c Data supporting this regimen come mainly from Europe and are based on the use of bismuth subcitrate and metronidazole (400 mg tid). This is the most commonly used second-line regimen.
- dData supporting this regimen come from Europe. Although the two 5-day courses of different drugs have usually been given sequentially, recent evidence suggests no added benefit from this approach. Thus 10 days of the four drugs combined may be as good and may aid compliance.
- e Data supporting this second- or third-line regimen come from Europe. This regimen may be less effective where rates of quinolone use are high. Theoretically, it may also be wise to avoid it in populations where Clostridium difficile infection is common after broad-spectrum antibiotic use.
The standard first-line therapy is a one week triple therapy consisting of proton pump inhibitors such as omeprazole and the antibiotics clarithromycin and amoxicillin [6,7]. Variations of the triple therapy have been developed over the years, such as using a different proton pump inhibitor, as with pantoprazole or rabeprazole [7].
The two most important factors in successful H. pylori treatment are the patient's close compliance with the regimen and the use of drugs to which the patient's strain of H. pylori has not acquired resistance. Treatment failure following minor lapses in compliance is common and often leads to acquired resistance to metronidazole or clarithromycin. To stress the importance of compliance, written instructions should be given to the patient, and minor side effects of the regimen should be explained. Resistance to clarithromycin and, to a lesser extent, to metronidazole is of growing concern. Clarithromycin resistance is less prevalent but, if present, usually results in treatment failure. Strains of H. pylori that are apparently resistant to metronidazole are more common but still may be cleared by metronidazole-containing regimens, which have only slightly reduced efficacy. Assessment of antibiotic susceptibilities before treatment would be optimal but is not usually undertaken because endoscopy and mucosal biopsy are necessary to obtain H. pylori for culture and because most microbiology laboratories are inexperienced in H. pylori culture. In the absence of susceptibility information, a history of the patient's (even distant) antibiotic use for other conditions should be obtained; use of the agent should then be avoided if possible, particularly in the case of clarithromycin (e.g., previous use for upper respiratory infection). If initial H. pylori treatment fails, one of two strategies may be used. The more common approach is empirical re-treatment with another drug regimen, usually quadruple therapy. The second approach is endoscopy, biopsy, and culture plus treatment based on documented antibiotic sensitivities. If re-treatment fails, susceptibility testing should ideally be performed, although empirical third-line therapies are often used.
Clearance of non-pylori gastric helicobacters can follow the use of bismuth compounds alone or of triple-drug regimens. However, in the absence of trials, it is unclear whether this outcome represents successful treatment or natural clearance of the bacterium
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