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CLINICAL MICROBIOLOGY AND VIROLOGY

Helicobacter pylori antibiotic resistance and [13C]urea breath test values

Vincenzo De Francesco, Angelo Zullo, Federico Perna, Floriana Giorgio, Cesare Hassan, Lucy Vannella, Francesca Cristofari, Carmine Panella, Dino Vaira, Enzo Ierardi

  • 1Section of Gastroenterology, Department of Medical Sciences, University of Foggia, Foggia, Italy
  • 2Gastroenterology and Digestive Endoscopy, ‘Nuovo Regina Margherita’ Hospital, Rome, Italy
  • 3Department of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy
  • Correspondence
    Vincenzo De Francesco
    vdefrancesco{at}alice.it

    • Journal of Medical Microbiology 2010; 59(5):588–591 · https://doi.org/10.1099/jmm.0.018077-0

    View at publisher PubMed

    Abstract

    A correlation between δ over baseline (DOB) values of the [13C]urea breath test (UBT) and Helicobacter pylori clarithromycin resistance has been reported, suggesting a possible predictive role of UBT in therapeutic outcome. However, available data are limited and conflicting. This study aimed to clarify this issue, assessing the possible relationship between H. pylori resistance towards different antibiotics (clarithromycin, metronidazole and levofloxacin) and UBT values. The data showed similar DOB values between susceptible and resistant strains for clarithromycin (46.9±32.3 vs 45.7±30.6; P=0.8), metronidazole (46.4±29.6 vs 47.4±37.9; P=0.8), and levofloxacin (45.0±30.2 vs 54.2±38.4; P=0.08). Likewise, comparable DOB values were observed between susceptible and multidrug-resistant strains (45.4±29.6 vs 54.8±44.8; P=0.1). In conclusion, our data failed to find a significant correlation between UBT values and H. pylori antibiotic resistance.

    INTRODUCTION

    It is widely accepted that antibiotic resistance is the main factor affecting the efficacy of therapy for Helicobacter pylori infection (Megraud, 2004). Currently, bacterial susceptibility towards different antibiotics is performed by using either an Etest on cultures or culture-free techniques, such as PCR (Owen, 2002). Undeniably, both culture and PCR-based methods have some limitations. Indeed, H. pylori is a quite fastidious bacterium so that the sensitivity of the culture may be reduced, even in expert hands (Zullo et al., 2003), whilst PCR-based techniques are available only in selected centres (De Francesco et al., 2006a, b, c,2007). Therefore, to have a non-invasive, widely accessible and cheap method for anticipating the presence of H. pylori resistance would be a definite advantage for the infection management in clinical practice. It has been suggested that the [13C]urea breath test (UBT) may predict clarithromycin resistance and, consequently, H. pylori eradication therapy outcome. In detail, a study reported a significant correlation between the δ over baseline (DOB) values of UBT and the presence of primary antibiotic resistance (Kawai et al., 2008). Moreover, increased DOB values have been observed in eradication failure patients harbouring clarithromycin-resistant strains as compared to those with susceptible strains. These data suggest that UBT may predict secondary clarithromycin resistance (Kao et al., 2005), although data are conflicting (Buzás & Széles, 2008). Other studies failed to confirm the correlation between DOB values and primary clarithromycin resistance – the key antibiotic for H. pylori therapy – suggesting the need of further studies on this topic (Zullo et al., 2008). We designed the present study with the aim of assessing whether DOB values are associated either with primary H. pylori resistance towards different antibiotics, as determined by culture and Etest, or with different point mutations of clarithromycin resistance, assessed by TaqMan real-time PCR.

    METHODS

    Patients.

    This was a post hoc analysis of a large study we recently published (Zullo et al., 2007). Briefly, the study population consisted of patients with dyspeptic symptoms and positive UBT, who were never previously treated for H. pylori infection. All patients underwent endoscopy with biopsies for histology (two samples from the antral gastric mucosa and two samples from the body gastric mucosa), and a rapid urease test (one sample from the antral gastric mucosa). Two further biopsies were taken for bacterial culture. Overall, data regarding 240 out of 255 patients enrolled in the previous trial were considered for the present post hoc study. Nine patients of the initial study were not considered because of a lack of levofloxacin resistance assessment, whilst six patients were excluded due to a lack of UBT values.

    [13C]UBT.

    All UBTs were carried out after an overnight fast. A baseline breath sample was obtained and then 75 mg [13C]urea with citric acid (1.5 g) was administered in water solution. Another breath sample was collected 30 min after administration of the test solution and the test was considered positive if there was a difference between the baseline sample and the 30 min sample that exceeded 4.5 parts per 1000 of 13CO2. All the breath samples were analysed using a single gas isotope ratio mass spectrometer (Finnigan) in the Department of Internal Medicine and Gastroenterology, University of Bologna. The accuracy of the UBT was previously validated with sensitivity and specificity values of 94.7 and 95.7 %, respectively (Vaira & Vakil, 2001).

    Culture and Etest.

    Biopsies collected for bacterial culture were streaked onto Columbia agar enriched with 5 % horse blood, containing 100 μg vancomycin ml−1, 200 μg trimethoprim ml−1, 10 μg polymixin B ml−1 and 10.7 μg nalidixic acid ml−1 to inhibit the growth of microbes other than H. pylori. The plates were incubated in a microaerobic environment at 37 °C for 7 days, and inspected daily from the third day. The isolates were identified by Gram stain, and by oxidase, catalase and urease tests. Suspensions from primary plates were prepared in sterile saline solution (0.9 % NaCl) at approximately 108 c.f.u. ml−1, an amount sufficient for performing the Etest. A blood agar plate (Muller–Hinton with 5 % horse blood but without any antibiotics) was streaked in three directions with a swab dipped into each bacterial suspension to produce a lawn of growth. An Etest strip (AB Biodisk) was placed onto a separate plate that was immediately incubated in a microaerobic atmosphere at 37 °C for 72 h. Isolated strains were tested for clarithromycin, metronidazole and levofloxacin resistance using >1, >8 and >1 mg l−1 as break point the MIC, respectively.

    TaqMan real-time PCR.

    For the present study, paraffin-embedded antral biopsy specimens were retrieved and used for clarithromycin-resistance assessment by TaqMan PCR. The technique was performed in blind in a single centre (Section of Gastroenterology, Department of Medical Sciences, University of Foggia). DNAs were extracted from the same antral samples used for histology using a NucleoSpin tissue kit (Macherey-Nagel) applied on paraffin-embedded sections (at least five sections of 10 μm thickness), which are largely accepted to constitute a reliable substrate for DNA analysis like fresh material (De Francesco et al., 2006a, b, c,2007). The A2142C, A2142G and A2143G point mutations were analysed by a novel method (TaqMan real-time PCR) that was first used for Mycobacterium tuberculosis detection, and then successively for H. pylori DNA sequencing on paraffin-embedded samples, as we have recently reported (De Francesco et al., 2007). Briefly, real-time PCR was performed in 96-well plates on the ABI Prism 7900HT sequence detection system (Applied Biosystems); data collection and analyses were carried out using the specific software (from the same provider). The final reaction volume (25 μl) was analysed in triplicate (three samples for each patient) and all experiments were repeated twice. Primers and probes for allelic discrimination are listed in previous works (De Francesco et al., 2006a, b, c,2007). The analysis yielded three major clusters corresponding to the three genotypes: homozygous wild-type, homozygous and heterozygous mutated type or heteroresistant status (combination of susceptible and resistant strains). The operator who performed all the PCR procedure was working blind of the culture results. In our experience, the variability between duplicates and triplicates within the same run or different runs was usually between 0 and 2 %, in agreement with that reported in other studies (Gerard et al., 1998).

    Statistical analysis.

    The statistical analysis was performed using Student's t-test for paired and unpaired data as appropriate. The results are expressed as means±sd. Differences were considered significant at the 5 % probability level. The analyses were performed with Statsoft 7.1 program for Windows XP.

    RESULTS AND DISCUSSION

    The baseline demographic characteristics are reported in Table 1⇓. Overall, 119 (49.6 %) patients were infected with an antibiotic-susceptible strain, whilst a bacterial resistance towards at least one of the tested antibiotics was present in the remaining 121 (50.4 %) patients (Table 2⇓). Overall, there were 39, 65 and 44 patients infected with either clarithromycin-, metronidazole- or levofloxacin-resistant strains, respectively, whilst 26 patients were infected with multidrug-resistant (double or triple drug) strains. By considering antibiotic resistance assessed by Etest, comparable DOB values were observed between susceptible and single-drug-resistant strains (45.4±29.6 vs 46.1±30.8; P=0.8). In detail, comparable values emerged between susceptible and resistant strains for clarithromycin (46.9±32.3 vs 45.7±30.6; P=0.8), metronidazole (46.4±29.6 vs 47.4±37.9; P=0.8) and levofloxacin (45.0±30.2 vs 54.2±38.4; P=0.08). Similarly, comparable DOB values were observed between susceptible and multidrug-resistant strains (45.4±29.6 vs 54.8±44.8; P=0.1).

    Table 1.

    Baseline, demographic and clinical characteristics of patients

    Table 2.

    Pattern of primary bacterial resistance towards different antibiotics

    In a subgroup of 39 patients showing clarithromycin resistance by culture, a TaqMan real-time PCR was performed to search for the three specific point mutations linked with clarithromycin resistance. A point mutation was detected in 37 out of these cases with a phenotypic clarithromycin-resistant strain. In detail the A2143G point mutation was observed in 22 patients, the A2142G point mutation in 10 patients and the A2142C point mutation in 5 patients. As shown in Table 3⇓, no significant difference in DOB values was observed between the three different clarithromycin-resistance point mutations. In the 19 patients (16 with a resistant strain and 3 with a susceptible strain) in whom bacterial eradication failed no significant modifications in DOB values emerged following therapy (56.4±38.2 vs 51.0±34.0; P=0.5). In detail, in infected patients with a resistant strain DOB values did not significantly change (58.6±42.0 vs 54.7±34.9; P=0.7), whilst a significant reduction was observed in those patients harbouring a susceptible strain (51.7±24.8 vs 31.0±22.8; P=0.01).

    Table 3.

    DOB values and different clarithromycin point mutations

    No significant difference emerged between the groups (P=0.3 for A2143G vs A2142G mutation genotype; P=0.6 for A2143G vs A2142C mutation genotype).

    Some studies have suggested that high DOB values are associated a high bacterial load in the stomach (Perri et al., 1998; Zagari et al., 2005; Cremonini et al., 2005), as well as with some H. pylori virulence factors, such as CagA (Zullo et al., 2004; Sarker et al., 2004). Moreover, in other studies increased DOB values were found in bacterial strains harbouring antibiotic resistance (Kawai et al., 2008; Kao et al., 2005). Our data failed to confirm a possible predictive role of UBT for bacterial resistance. Indeed, we observed similar DOB values between patients infected with either susceptible or resistant H. pylori strains towards the different antibiotics tested. Clarithromycin resistance is regarded as the key factor involved in H. pylori therapy failure, and some studies found that the main three different point mutations implicated in such resistance are associated with different MIC values in vitro (Owen, 2002; Megraud, 2004). We previously found that the A2143G point mutation, but not the other two point mutations, significantly reduced the eradication rate following therapy (De Francesco et al., 2006a). The present study found comparable DOB values among strains with the three different point mutations of clarithromycin resistance. Therefore, on the basis of UBT values it does not seem to be possible to predict the presence of different clarithromycin-resistance point mutations and, in turn, to anticipate the therapy success probability. Therefore, the bacterial urease activity, which correlates with DOB values, seems to mainly depend on the overall bacterial load rather than on the presence of antibiotic resistance. In conclusion, our data based on both phenotypic and genotypic testing showed that presence of primary H. pylori resistance towards different antibiotics is not associated with different UBT values as compared to susceptible strains.

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