In vitro activity of fluoroquinolone and the gyrA gene mutation in Helicobacter pylori strains isolated from children

Helicobacter pylori is associated with the pathogenesis of chronic gastritis, peptic ulcers and gastric cancer (National Committee for Health, 1994). A combination of a proton pump inhibitor and two antibiotics has been widely used as the standard regimen for H. pylori eradication. In this triple eradication therapy, amoxycillin, clarithromycin and metronidazole are most often used (Lind et al., 1999). Studies in adults have demonstrated that triple therapies with two out of these three antibiotics usually achieve an eradication rate above 80 % (European Helicobacter pylori Study Group, 1997). Although H. pylori has retained susceptibility to amoxycillin, strains resistant to clarithromycin and metronidazole have been frequently found worldwide (Glupczynski et al., 2001). In our recent study in Japanese children, primary resistance to clarithromycin and metronidazole was at the level of 29 and 24 %, respectively (Kato et al., 2002). In Europe, the rates of primary resistance to clarithromycin and metronidazole ranged between 0 and 22 % and between 16 and 61 %, respectively (Megraud et al., 1999). Clarithromycin and metronidazole resistance is thought to be the major cause of H. pylori eradication failure (Adamek et al., 1998). However, efficient second-line therapy has not yet been established. Although fluoroquinolones cannot usually be used in children, these antibiotics have been reported to be effective against H. pylori strains isolated from adults (Boyanova et al., 2000). In H. pylori strains isolated from children, however, there are no reports on fluoroquinolone susceptibility. In the present study, we examined the in vitro susceptibility to levofloxacin and ciprofloxacin of H. pylori strains isolated from Japanese children. Furthermore, mutations in the gyrA gene, which encodes subunit A of DNA gyrase, were investigated for fluoroquinolone resistance. Bacterial strains.
A total of 55 H. pylori strains were isolated from gastric biopsies of 55 paediatric patients and studied. The patients (age range 517 years) underwent upper gastrointestinal endoscopy and gastric biopsy at Tohoku University Hospital because of gastrointestinal symptoms. None of the patients had a previous history of H. pylori eradication therapy or of the use of fluoroquinolone, clarithromycin and metronidazole. Informed consent was obtained from parents of all patients before inclusion in the study.

Antibiotic susceptibility testing.
Using the Etest according to the manufacturer's recommendations (AB Biodisk), the MICs of amoxycillin, clarithromycin, metronidazole, levofloxacin and ciprofloxacin were determined under microaerobic conditions (5 % CO₂, 5 % O₂, 90 % N₂) for 72 h on MuellerHinton agar (Eiken) supplemented with 5 % defibrinated sheep blood (Sigma). H. pylori was considered to be resistant to fluoroquinolones (levofloxacin and ciprofloxacin), amoxycillin, clarithromycin and metronidazole when the MICs were greater than 1, 0.5, 0.5 and 8 mg l¹, respectively (Wang et al., 2001). H. pylori NCTC 49503 was studied as the control organism.

PCR amplification and nucleotide sequence.
Genomic DNA of fluoroquinolone-resistant strains was extracted by a method described elsewhere (Fujimura et al., 2002). Oligonucleotide primers gyrAPF1 (5'-ATGCATGAATTAGGTCTTACT-3') and gyrAP2 (5'-TTCTTCAC TCGCCTTAGTCAT-3'), flanking the gene fragment encoding the fluoroquinolone-resistance-determining region, were designed from the H. pylori gyrA gene. These primers were modified versions of the primer pair used by Wang et al. (2001). PCR was performed with 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, and extention at 72 °C for 2 min. PCR products for sequencing were purified with a PCR Kleen Spin Column (Bio-Rad). PCR templates of all strains were sequenced directly on both strands by the BigDye Terminator Cycle Sequencing method using an ABI PRISM 377XL sequencer (Applied Biosystems). The sequences were compared with the published sequence of the H. pylori gyrA gene (GenBank accession no. L29481).

The MICs of antibiotics are shown in Table 1. Of the 55 strains tested, 12 (21.8 %) were resistant to clarithromycin. Of these 12 strains, 11 (91.7 %) were susceptible to both levofloxacin and ciprofloxacin. On the other hand, three strains (5.5 %), including one clarithromycin-resistant strain, were resistant to both levofloxacin and ciprofloxacin. The MICs for fluoroquinolone of the resistant strains were 1.5 mg l¹ in one strain and >32 mg l¹ in two. In 52 fluoroquinolone-susceptible strains, the MICs for levofloxacin and ciprofloxacin were similar (differences of < 2 doubling dilutions). Resistance to metronidazole and amoxycillin was demonstrated in 9.1 (five strains) and 0 % of the strains, respectively. All of the five metronidazole-resistant strains were susceptible to these fluoroquinolones.

Table 1. MICs (mg l1) of antibiotics for 55 H. pylori strains isolated from paediatric patients

In three strains resistant to fluoroquinolones, sequence analyses of the gyrA gene showed the missense mutations including point mutations of G271A (Asp to Asn), G271T (Asp to Tyr) and A272G (Asp to Gly) (Table 2). These three point mutations indicated mutations of the codon Asp91 in the fluoroquinolone-resistance-determining region of the DNA gyrase (Table 2).

Table 2. Mutations of the gyrA gene from fluoroquinolone-resistant H. pylori isolated from biopsy samples TH 1, 2 and 3 were isolated from children in this study, and UC reference strains were from adults. X, Presence of indicated mutation.

Clarithromycin resistance frequently causes failure of H. pylori eradication. In Japan, the rate of primary resistance to clarithromycin was 29 % in H. pylori strains from children (Kato et al., 2002) and 13 % in those from adults (Kato et al., 2000). The eradication rates of the clarithromycin-resistant strains were significantly lower than those of the susceptible strains (Kato et al., 2002; Murakami et al., 2002). In clarithromycin-resistant H. pylori strains, it has been shown that an increased dose of clarithromycin does not improve the eradication rate (Murakami et al., 2002). However, the present study demonstrated that levofloxacin and ciprofloxacin have in vitro anti-H. pylori activities against 91.7 % of clarithromycin-resistant strains. In animal studies (Takada et al., 1994), however, toxic effects of fluoroquinolones, such as irreversible cartilage and skeletal abnormalities, have been reported. Although such adverse effects have not been documented in humans, fluoroquinolones are generally contra-indicated in patients less than 18 years old. Gendrel et al. (2003) have stressed that it is important to continue the policy of second-line use in children only when earlier treatment is unsuccessful or when other antibiotics approved for children cannot be used. Although the available information on the paediatric use of levofloxacin is limited, levofloxacin has better tissue permeability than ciprofloxacin and the incidence of adverse effects is low (Croom & Goa, 2003). Furthermore, levofloxacin is reported to be effective as a second- or third-line H. pylori therapy for adult patients in whom eradication with first-line therapy failed (Nista et al., 2003; Watanabe et al., 2003). Considering these facts, we believe that levofloxacin should be considered as one of the antibiotics used for second- or third-line H. pylori eradication therapy in children.

Moore et al. (1995) reported four mutations of the A subunit of the DNA gyrase (GyrA) at amino acid 87 (Asn to Lys), 88 (Ala to Val), 91 (Asp to Gly, Asn or Tyr) and a double substitution at 91 and 97 (Ala to Val) in H. pylori strains with secondary resistance to ciprofloxacin. Moreover, in one in vitro study with the serial passage method, GyrA mutation at amino acid 91 (Asp to Ala, Gly, Asn or Tyr) was also reported in the ciprofloxacin-resistant strains (Wang et al., 2001). Our primary resistant strains showed three point mutations at amino acid 91 (Asp to Tyr, G271T; Asp to Asn, G271A; Asp to Gly, A272G). To our knowledge, this is the first report concerning the gyrA mutation of H. pylori strains isolated from children.

In Iranian children, resistance to ciprofloxacin was detected in 20 % of H. pylori strains. Falsafi et al. (2004) reported that Iranian children take ciprofloxacin for treatment of dysentery and salmonella infections. Our study results indicate that the rate of primary fluoroquinolone resistance was low (5.5 %) because fluoroquinolones were not used in children in Japan. In European children, primary resistance of H. pylori to clarithromycin, metronidazole and ciprofloxacin was in 9.7, 28.6 and 3.9 % of the strains, respectively (Boyanova et al., 2000). Furthermore, in one study with participants over 10 years, the overall rates of H. pylori strains resistant to clarithromycin, metronidazole and ciprofloxacin were 19.0, 30.6 and 9.6 %, respectively (Cabrita et al., 2000). On the other hand, Heep et al. (2000) noted that secondary H. pylori resistance to clarithromycin and metronidazole after eradication failure was frequently observed in 75 and 58 % of the strains, respectively, but resistance to ciprofloxacin was observed in only 9 % of the strains. In this way, in Japan and parts of Europe, fluoroquinolones are not approved for other infectious diseases in children. Among the fluoroquinolones, only norfloxacin is approved for some infectious diseases in Japanese children. However, it is extremely rare that norfloxacin is used in clinical practice in children because ß-lactams and macrolides are commonly chosen for the treatment of common bacterial infections. Therefore, it is thought that the rate of resistance to fluoroquinolones in H. pylori isolated from children is low. There is a little information about resistance to fluoroquinolones in adults, but it is very likely that these antibiotics are effective in Japanese children. At present, it is thought that fluoroquinolones continue to be active against most H. pylori strains. It remains unclear whether primary resistance to fluoroquinolones in children indicates spontaneous resistance or acquisition of the resistant strain. In either event, it is suggested that it is difficult for H. pylori to acquire secondary resistance to fluoroquinolones.