Ltr_helicobater Pylori Antibiotic Susceptibility And Resistance

Helicobater Pylori Antibiotic Susceptibility and Resistance David Yong Seng Chai1 1

Chung Ling High School Penang, Malaysia

Following the discovery of Helicobater Pylori (H. Pylori) in 1982, various antibiotics have been used in an effort to eradicate it from the human body. Treatment to eradicate H pylori in patients with a proved ulcer is important and benefits the patient and society (1). Initial efforts employed the use of 5-nitroimidazoles, mainly tinidazole and metronidazole (2), agents that are frequently used in the treatment of anaerobic bacteria and selected protozoan infections (3). The following years saw the use of other drugs, namely a macrolide compound, clarithromycin, and a β-lactam compound, amoxicillin. These drugs are frequently employed in a triple or quadruple combination including a Proton Pump Inhibitor (PPI) or H2-receptor antagonist (4). This has been backed up by the MACH-1 (5) and MACH-2 (6) studies that provide data that the combination of a a PPI together with clarithromycin and metronidazole or clarithromycin and amoxicillin represent a highly effective treatment regimen. Several equally effective regimens, that last from 7 to 21 days, are also available, but even the best fail in 5-20% of patients (7). These failures occur do to H. Pylori’s resistance towards antibiotics, which is growing prevalent, as supported by various data sources that will be subsequently provided. Treatment failure due to resistance result in the patient being forced to undertake other regimens with different key antibiotics. This in turn causes various adverse short and long term effects towards the human body (8). This literature aims to review the susceptibility of H. Pylori towards various antibiotic groups, and in light of growing prevalence of resistance, the resistance mechanisms involved. 5-Nitroimidazoles 5-Nitroimidazoles are prodrugs and need to be activated within the target cell. They possess a very low reduction potential and are activated by a one-electron reduction process. This reduction leads to the generation of reactive products, primarily free radicals, which damage subcellular structures and cause damage coupled with lethal mutations in the DNA (9). Some of the reduction products are mutagenic and their formation can lead to the generation of secondary free radicals, which are also damaging. With the reduction of the drug, a concentration gradient is formed that facilitates the diffusion of particular 5-nitroimidazoles into the cell. (9). Only anaerobes have the reductive capacity to activate 5-nitroimidazoles, whereas aerobic bacteria and mammalian cells do not. H. Pylori’s susceptibility to 5-nitroimidazoles is an indication that the microaerophile has an atypical metabolism.

H. Pylori possesses an enzyme, pyruvate:flavodoxin oxidoreductase (porGDAB) (10) that catalyzes the formation of acetyl-CoA from pyruvate. pyruvate:flavodoxin oxidoreductase is important because in anaerobes, the related enzyme system, pyruvate:ferridoxin oxidoreductase, is central to the activation of 5-nitroimidazoles (9). Hughes et al. was able to proof through indirect evidence that pyruvate:flavodoxin oxidoreductase of H. Pylori is able to activate metronidazole (11) (12). Although they showed pyruvate:flavodoxin oxidoreductase is the essential activator of H. Pylori, they were unable to determine its exact contribution. After understanding the activation process, it is noted here that the commonly used 5Nitroimidazoles drug is Metronidazole. Metronidazole is a generic drug sold as Flagyl, MetroGel and Protostat that is prescribed during conditions where anaerobic bacteria or protozoan parasites are suspected. It is frequently used as a key drug in many H. Pylori treatment regimens. After being activated by pyruvate:flavodoxin oxidoreductase, Metronidazole subsequently activates a genes known as rdxA nitroreductase (rdxa) (13). rdxA is responsible for coding nitroreductase enzymes that allow H. pylori to break down organic nitrogen compounds. The enzyme also happens to convert metronidazole to hydroxylamine, which damages DNA, proteins and other macromolecules and kills the bacteria. Therefore the bacterium changes a harmless chemical into a lethal drug which is used against it. Metronidazole is highly effective against clinical strains of H. Pylori with a Minimum Inhibitory Concentration (MIC) of <4mg/ml (14). The recommended treatment dosage is 800-1500 mg orally daily for several days in combination with other drugs. Its adverse effects are also minimal and are generally well tolerated with appropriate use. Minor side effects include nausea, headaches, loss of appetite, a metallic taste, and rashes. Serious side effects of metronidazole are rare, including seizures and damage of nerves resulting in numbness and tingling of extremities. Alcohol must be avoided following the usage of Metronidazole for 48 hours. Pyruvate:flavodoxin oxidoreductase (porGDAB) has been implicated in 5-nitroimidazole resistance (15). There is putative evidence that it can be deactivated, thus causing Metronidazole to remain dormant and unactivated. However further studies are required to provide solid proof. Besides that, Smith and Edwards (16) identified NADH oxdase activity as being associated with metronidazole resistance. NADH oxidases are enzymes that reduce molecular oxygen to hydrogen or water. There was a threefold greater NADH oxidase activity in cytosolic fractions from metronidazole-susceptible isolates compared with resistant isolates. Again, this only provides indirect potential mechanisms of resistance. The work of Hoffman et al. has provided invaluable insights into understanding Metronidazole resistance. On the basis of substantial molecular evidence, they reported that mutations in the gene encoding for an oxygen-insensitive nitroreductase (rdxA) lead to increased resistance due to the inactivation of nitroreductase (17). Another study from the same group established that 5-nitroimidazole resistance in H. Pylori strain ATCC 43504 was due to an insertion sequence (a mini-IS605) and deletions in the rdxA gene

(18). More importantly, this group noted no mutation in this strain compared with a susceptible strain in other putative "resistance" genes (catalase [katA], superoxide dismutase [sodB], flavodoxin [fldA], ferridoxin [fdx], pyruvate:flavodoxin oxidoreductase [porGDAB], and RecA [recA]). Furthermore, Tankovich et al. (19) reported four mechanisms of rdxA inactivation in clinical isolates from France and North Africa: frameshift mutations, missense mutations, deletion of bases, and the presence of an insertion sequence (mini-IS605). This, however, is not the whole picture yet. Jenks et al. (20) found metronidazoleresistant isolates of H. Pylori without apparent mutations in rdxA, supporting the belief that additional mechanisms of 5-nitroimidazole resistance must be present in some isolates of H. pylori. This conclusion has been supported by Kwon et al. (21) who indicated that in addition to rdxA, mutations in a number of genes may contribute to 5nitroimidazole resistance. Two other studies by Kwon et al. (21) also establish that in addition to rdxA, ferredoxinlike protein (fdxB) and an NAD(P)H flavin oxidoreductase (frxA) can contribute to the activation of metronidazole in H. Pylori and that resistance may occur through mutations in these genes (22) (23). They observe that the inactivation of fdxB, frxB, and rdxA result in different levels of metronidazole resistance in H. Pylori. Jin-Yong Jeong et al. (13) on the other hand provide a different set of findings. They conclude that resistance of Metronidazole requires inactivation of rdxA alone or of both rdxA and frxA, depending on bacterial genotype, but rarely, if ever, inactivation of frxA alone. At this junction, it can be speculated that there are various systems that might contribute to metronidazole resistance, though rdxA is the most important and major one. The prevalence of H pylori resistance to metronidazole varies from 20% to 40% in Europe and the USA. The prevalence is much higher in developing countries (50–80%), for example Mexico (76.3%). In contrast, the prevalence rate is quite low in Japan (9– 12%) (24). In the European multicentre study, the global resistance rate to metronidazole is 33.1% (95% CI 7.5–58.9) with no significant difference between the North (33% (95% CI 7.1– 69.2)) and South (40.8% (95% CI 27.3–54.3)) but a significantly lower prevalence in Central and Eastern parts (29.2% (95% CI 17.9–41.5)) (p<0.01). The global resistance rate shows a slight increase in comparison with that of a previous study carried out seven years earlier in Europe. When risk factors are studied, past use of metronidazole, which is common in tropical countries for parasitic diseases, is once more involved. In developed countries, most studies have reported a higher resistance rate in women than in men, probably due to the use of nitroimidazole drugs to treat gynaecological infections (25) .The use of nitroimidazole for dental infections may also add to selection pressure (26). In the USA there were no marked regional differences.

Macrolides Macrolides are a group of drugs whose activity stems from the presence of a macrolide ring, a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. Macrolides belong to the polyketide class of natural products. A common macrolide group used in the eradication of H. Pylori is clarithromycin. Along with metronidazole, clarithromycin is a key antibiotic in many treatment regimes. An intake of up to 500mg, 3 times a day are normally prescribed. It possesses some common gastrointestinal side-effects, diarrhea, nausea, abdominal pain, vomiting and facial swelling. Less common side-effects include headaches, dizziness/motion sickness, rashes, alteration in senses of smell and taste, including a metallic taste that lasts the entire time one takes it. Dry mouth, anxiety, hallucinations, and nightmares have also been reported. In more serious cases it has been known to cause jaundice, other liver disorders, and kidney problems including kidney failure. Clarithromycin may cause false positives on urine drug screens for cocaine. Clarithromycin works by penetrating into the bacterial cells and binding to the ribosomes. The target is the domain of the 23S rRNA, the peptidyl transferase loop in domain V. Binding of domain V leads to an interruption of protein elongation, causing the bacteria to be unable to synthesize protein, subsequently leading to death. Goldman et al. have shown that clarithromycin, its parent compound erythromycin, and its 14-hydroxy metabolite have the tightest binding interaction for a macrolide-ribosome complex (27). Resistance by efflux does not occur in H. Pylori. Bina et al. reported that independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19 antibiotics. These results, suggest that active efflux does not play a role in the intrinsic resistance of H. pylori to antibiotics. Another well-known mechanism of macrolide resistance that involves methylation of adenine residue has also not been found in clarithromycin resistant H. pylori strains (28) (29). Versalovic et al. on sucessfully demonstrate that point mutations, adenine to guanine, in two positions, 2142 (A2142G) and 2143 (A2143G), are responsible for clarithromycin resistantance. These mutations are associated with a lack of macrolides to isolated ribosomes, where the amount of bound antibiotics increase proportionally with the amount of purified ribosomes from the susceptible strain but not from the resistant strain (31). Other than transition mutations at points 2142 and 2143, which were found in clinical specimens, other mutations have been described. Debets-Ossenkop et al. were able to generate other mutataions (A2143C, A2142T, A2143T) in vitro. However, these mutant

strains were unstable and had a reduced growth rate (32). the mutant A2143T had an MIC of only 0.5 mg/liter. Transitional mutation at positions 2515, 2116 and 2144 have also been discovered byHsieh PF et al. and Hultén et al., with the mutations being described ashomozygous (28) (33). Simultaneous mutations A2115G and G2141A described by Hulten et al. have never been found again (28). The impact of specific mutations on the MIC has also been studied. In most studies, A2142G mutations were more significantly present in isolates with MICs of >64 mg/liter. H. pylori contains two 23S rRNA operons (35) that are known to undergo resistance mutations. It is understood that mutations occur spontaneously and are selected after exposure to the drug. The presence of a low number of bacterial cells with a given point mutation has been shown within a population of otherwise susceptible bacteria, even though the patient was not supposed to have previously consumed macrolides. The frequency of mutations leading to spontaneous resistance of clarithromycin has been found to be in the range of 3.2 × 10−7 to 6 × 10−8 in vitro (36). However, the figure is different in vivo due to higher bacterial mutation rates. Stability of clarithromycin resistance has also been evaluated by a disc diffusion test and confirmed by the plate dilution method. Among the 20 clarithromycin-resistant isolates, nine (45%) reverted to be sensitive after 2–5 subcultures on drug-free agar. The findings in this study indicate that the incidence of clarithromycin-resistant H. pylori in untreated dyspeptic patients is low. Cross-resistance that occurs between macrolides and resistance to clarithromycin in some strains is totally reversible (37). The stability of mutants A2142G and A2143G has also been questioned, since resistance usually has a biological cost (38). A multicentre survey conducted in Europe saw adults in Sourthern Europe possessing a resistance prevalence of more than 20% while Northern Europe was around 5%. Children, had a prevalence ranging from 12.4% to 23.5%. The global primary resistance rate for clarithromycin was 9.9%, probably due to resistance of H. pylori to macrolides being chromosomally mediated and essentially transmitted vertically to the bacterial descendants. Clinical trials in USA showed prevalence rates of 10 to 15% while the Middle East had a rate of 17%. Through the data of studies conducted over the years, a trend of increasing prevalence can be clearly deduced. The essential risk factor for clarithromycin resistance is previous consumption of macrolides (39). In Japan, clarithromycin consumption for other purposes multiplied by four between 1993 and 2000, and this led to a fourfold increase in clarithromycin resistance during H. Pylori treatment (40). For most resistance cases, the failure of a clarithromycin-based therapy, a clarithromycin strain is isolated in as many as 60% of the cases. Statistics show that when a

clarithromycin-based triple therapy is given to a patient harboring a resistant strain, the chance of success decreases by more than 50%. β-lactam β-lactam antibiotics are a broad class of antibiotics that include penicillin derivatives, cephalosporins, monobactams, carbapenems, and β-lactamase inhibitors. The group includes any antibiotic agent that contains a β-lactam nucleus in its molecular structure. They are the most widely-used group of antibiotics. The only β-lactam used to treat H. Pylori infections is amoxicillin. Amoxicillin has a very low MIC against H. Pylori, approximately <0.03 μg/ml, and is frequently included in most current therapeutic regimens. β-lactam antibodies work by inhibiting the synthesis of the peptidoglycan layer and crosslinkage between the linear peptidoglycan polymer chains of bacterial cell walls. The peptidoglycan layer is important for cell wall structural integrity. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by transpeptidases known as penicillin-binding proteins (PBPs). β-lactam antibiotics are analogues of D-alanyl-D-alanine - the terminal amino acid residues on the precursor NAM/NAG-peptide subunits of the nascent peptidoglycan layer. The structural similarity between β-lactam antibiotics and D-alanyl-D-alanine facilitates their binding to the active site of penicillin-binding proteins (PBPs). The βlactam nucleus of the molecule irreversibly binds to (acylates) the Ser403 residue of the PBP active site. This irreversible inhibition of the PBPs prevents the final crosslinking or transpeptidation of the nascent peptidoglycan layer, disrupting cell wall synthesis. Under normal circumstances peptidoglycan precursors signal a reorganisation of the bacterial cell wall and, as a consequence, trigger the activation of autolytic cell wall hydrolases. Inhibition of cross-linkage by β-lactams causes a build-up of peptidoglycan precursors, which triggers the digestion of existing peptidoglycan by autolytic hydrolases without the production of new peptidoglycan. As a result, the bactericidal action of βlactam antibiotics is further enhanced. Treatment with amoxicillin has a normal dosage of 500mg to 875 mg. Side effects which may occur due to amoxicillin include diarrhea, dizziness, heartburn, insomnia, nausea, itching, vomiting, confusion, abdominal pain, easy bruising, bleeding, rash, and allergic reactions. Individuals who are allergic to antibiotics in the class of cephalosporins may also be sensitive to amoxicillin. Previously, amoxicillin resistant strains have only been found in clinically cultured strains through serial passages in increasingly subinhibitory concentrations of amoxicillin (36). Theses strains exhibited MICs of between 0.25 to 0.5 μg/ml, up to 100 times of the most susceptible strains. Resistance towards amoxicillin isolated from patients however has not been discovered until quite recently.

Dore et al. reported the occurrence of 17 amoxicillin resistant strains isolated from patients in Italy and the United States (36). These strains showed pretreatment amoxicillin resistance with MICs of >256 μg/ml and resulted in a marked reduction in treatment efficiency. The amoxicillin resistance phenotype was unstable and lost after storage at −70°C, but plating these strains on amoxicillin gradient plates could restore resistance (41). A H. pylori strain has also been isolated in The Netherlands, in which, in contrast, the amoxicillin resistance remained stable after repeated cycles of freezing and culture (42). However, the MIC for this strain was 8 μg/ml, which is relatively low. Amoxicillin resistance could be transferred from the resistant strain to a susceptible one by natural DNA transformation at a frequency of 10−5 bacteria, leading to the production of stable resistant transformants for which the MICs were up to 400 times greater than the value for the susceptible strain and similar to that for the naturally resistant strain. Amoxicillin resistance also leads to cross-resistance agains other β-lactam antibodies (43). In general, amoxicillin MICs of >32 μg/ml were considered significantly resistant while those with an MIC of ≈2 μg/ml were considered low-level. Resistance to β-lactam antibiotics by gram-negative bacteria is most commonly due either the production of β-lactamase or the altering of penicillin-binding proteins (PBPs). The first method, production of β-lactamase, wither chromosomally encoded or plasmid mediated works by breaking open the β-lactam ring of the antibiotic, rendering the antibiotic ineffective. In H. pylori, amoxicillin resistance could not be attributed to the expression of a β-lactamase, since β-lactamase activity was not detected by the chromogenic cephalosporin nitrocefin assay in any of the resistant strains (44). This is due to the fact that there are no β-lactamase homolog genes in the two H. Pylori strains sequenced. This leaves the other mode of resistance, alterations in the PBPs combined with decreased permeability of the antibiotic into the bacterial cell wall. PBPs are a set of enzymes found in the cytoplasmic membrane of bacteria that are involved in the terminal stages of peptidoglycan biosynthesis (45). They are integral components in the determination and maintenance of cellular morphology and the target proteins for penicillin and other β-lactam antibiotics. Covalent binding of the β-lactams to specific PBPs in susceptible organisms results in the inability of the bacterium to build a complete cell wall and leads to rapid cell lysis and death. In this case, alterations in the PBPs, which affect the ability of the β-lactams to bind, confers increased resistance to amoxicillin. Initial findings showed that in H. Pylori, there existed three major PBPs (PBP 1, 2, 3) with molecular masses of 66, 63, and 60 kDa (PBP 1, 2, and 3, respectively) (46). Dore et al. (47) and Krishamurthy et al. (48) in two different studies, both found a previously undetected PBP-D (and later known as PBP-4), with a molecular mass of 32 kDa which was a cause of amoxicillin resistance. Besides that, comparative analysis of the PBP profiles generated from bacterial membranes of the susceptible strain and of the resistant strain revealed a significant decrease in labeling of PBP 1 by biotinylated amoxicillin in

the amoxicillin-resistant strain. Kusters et al. (49) also supported this by showing that point mutations in the PBP 1A gene were associated with stable amoxicillin resistance, and that this resistance could be transferred to a susceptible strain by natural DNA transformation. In several multicentre studies, resistance to amoxicillin has consistently hovered around null and 2%. Even in a large European multicenter trial, no amoxicillin resistance was found among the 485 H. pylori isolates tested (50). However, some alarming rates have been reported in certain studies, 26% in Italy (51), 29% in Brazil (52) and even 41% in China (53). This may be due to testing methods or epidemiologic differences. Nevertheless, the emergence of high-resistance strains with MICs of > 256 μg/ml represents a major threat to this effective H. pylori eradication method and requires constantly monitoring of H. pylori susceptibility towards amoxicillin. Double Resistant Strains As Clarithromycin and metronidazole are the antibiotics most frequently used with amoxicillin, it is interesting to note a generation of double resistant strains. Their frequency has exceeded previous forcasts. their proportion among the total number of strains is 0.8–9.1% in Europe, 2–3%, in Asia and much higher in developing countries such as Mexico (18%). If the failure of a therapy using both clarithromycin and metronidazole occurs, then up to 50% of strains may harbour double resistance (54). Conclusion The three groups of antibiotics reviewed above are currently the most effective and commonly used antibiotics in H. Pylori eradication treatment regimes. They are frequently used together with PPIs to triple or quadruple regimens which will be addressed in the next section, current treatment regimes. Although these antibiotics possess good susceptibility, H. Pylori’s global resistance prevalence towards them is alarming. Taking into account the trend of increasing resistance that has been shown worldwide, therapy regimes failure rates will increase even further until the 30% threshold even for previously highly effective combinations. Furthermore, the continued intake of antibiotics poses evident, if not serious, adverse effects towards human health. Therefore, it is fair to conclude that new alternatives need to be discovered and researched in the near future.

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