Armelle Ménard

Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in Helicobacter pylori

ABSTRACT The main cause of failure of Helicobacter pylori eradication therapy is resistance to clarithromycin. The resistance is due to three point mutations in two positions on the 23S rRNA (A2142C, A2142G, and A2143G). Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR that included a simultaneous detection of the amplicons by hybridization of two probes labeled with LC-Red and fluorescein by using the fluorescence resonance energy transfer (FRET) technology and melting curve analysis with the LightCycler thermocycler. The assay was first applied successfully on reference strains, reference plasmids, and H. pylori-negative biopsies. Biopsies from 200 patients having failed a first eradication attempt and for whom the H. pylori strain was available were then tested with the new assay. A result was obtained in 199 cases; a single genotype was detected in 157 cases, two genotypes were detected in 41 cases, and three genotypes were detected in one case. There were, in total, seven discrepancies between the real-time PCR and the phenotypic method of determination of clarithromycin susceptibility, and in an additional four cases the two phenotypic methods were in disagreement. PCR-restriction fragment length polymorphism was applied to a sampling of biopsies, including all of the cases with multiple genotypes and all the cases with discrepant results. Finally, in four cases with discrepant results, the real-time PCR detected the resistant population at a concentration so low that it could not be detected by the phenotypic method, while in three cases other mutations could be involved. This assay had an accuracy at least as satisfactory as that of the phenotypic tests and could be performed within 2 h, allowing it to be used before the administration of therapy in the case of a first H. pylori eradication.

Arcobacter butzleri: Underestimated Enteropathogen

Molecular methods applied to 2,855 strains of Campylobacter-like organisms received from a surveillance network of Campylobacter infections in France identified 29 Arcobacter butzleri infections. This species ranks fourth for Campylobacteraceae isolation and appears to have the same pathogenic potential as the other species in the genus.

Association of Helicobacter species with hepatitis C cirrhosis with or without hepatocellular carcinoma

Background and aims: Recent studies have suggested that bacterial coinfection with Helicobacter species in patients already infected with hepatitis C virus (HCV) could be involved in the development of cirrhosis and hepatocellular carcinoma (HCC). A retrospective cross sectional study was performed in order to explore the association between Helicobacter species and HCV associated liver diseases. Methods: The presence of Helicobacter species was tested by polymerase chain reaction on liver samples from four groups of patients. Results:Helicobacter 16S rDNA was found in only 4.2% of liver samples from control patients (n = 24) and in 3.5% of liver samples from patients with non-cirrhotic chronic hepatitis C (n = 29) while it was found in 68.0% of liver samples from patients with HCV positive cirrhosis without HCC (n = 25) as well as in 61.3% of cirrhotic liver samples from patients with HCV positive cirrhosis and HCC (n = 31). In addition, when the HCC tumour tissue was tested (n = 21), 90.5% of samples were positive. DNA from Helicobacter pylori- and Helicobacter pullorum-like organisms was found. Conclusions: There is an association between the presence of Helicobacter species DNA in the liver and hepatitis C cirrhosis, with or without HCC. Indeed, the presence of these bacteria could be the result of structural changes in the liver. Alternatively, Helicobacter species could be a co-risk factor in HCV chronic liver diseases. This result warrants prospective studies to determine the possible causal role of these bacteria in the progression of chronic hepatitis C.

Increased macrolide resistance of Mycoplasma pneumoniae in France directly detected in clinical specimens by real-time PCR and melting curve analysis

OBJECTIVES Mycoplasma pneumoniae is a common aetiological agent of community-acquired respiratory tract infections for which macrolides are the treatment of choice. In France, only two macrolide-resistant isolates were reported in 1999. In contrast, several recent data reported that macrolide-resistant M. pneumoniae isolates have been spreading since 2000 in Japan. Mutations A2058G (Escherichia coli numbering), A2058C, A2059G, A2062G, C2611A and C2611G in domain V of the 23S rRNA gene were associated in vivo or in vitro with this resistance. The aim of this study was to determine whether macrolide resistance of M. pneumoniae is emerging in France. PATIENTS AND METHODS We developed a duplex real-time PCR for the detection of the six 23S rRNA mutations associated with macrolide resistance in M. pneumoniae and a simplex real-time PCR for the identification of the A2058G mutation, the most common one. Both methods rely on fluorescence resonance energy transfer coupled to melting curve analysis and are directly applicable to clinical samples. The duplex real-time PCR assay, first validated on 40 genetically characterized M. pneumoniae strains, was then applied directly on 248 French respiratory tract clinical samples. RESULTS Among M. pneumoniae-positive specimens collected before 2005, no macrolide-resistant M. pneumoniae isolate was detected. In contrast, among 51 samples collected between 2005 and 2007, five (9.8%) yielded a resistant genotype, suggesting a recent increase in macrolide-resistant M. pneumoniae isolates in France. CONCLUSIONS The epidemiological monitoring of macrolide resistance in this species has become necessary in France and Europe, and will be made easier by using these PCR assays.

Evaluation of the Association of Nine Helicobacter pylori Virulence Factors with Strains Involved in Low-Grade Gastric Mucosa-Associated Lymphoid Tissue Lymphoma

ABSTRACT Helicobacter pylori has been associated with the development of two malignant diseases: gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Although the cag pathogenicity island, especially the cagA gene, has been linked with adenocarcinoma, few data concerning H. pylori pathogenic factors involved in low-grade gastric MALT lymphoma are available. The goal of this study was to analyze the prevalence of and correlation between genes coding for seven H. pylori virulence factors (cagA, cagE, vacA, iceA, babA, hopQ, and oipA) and two novel adhesins (sabA and hopZ) by comparing a collection of 43 H. pylori strains isolated from patients with low-grade gastric MALT lymphoma to 39 strains isolated from age-matched patients with gastritis only. Our results show that taken individually, none of the nine genes tested can be considered associated with MALT strains and allow us to conclude that MALT pathogenesis is not linked with more proinflammatory H. pylori strains. We demonstrated that in patients infected with strains harboring the iceA1 allele, sabA functional status, and hopZ “off” status, the odds of developing a MALT lymphoma were 10 times higher. However, the low prevalence of such strains (10 of 43 MALT strains) renders this triple association a low-sensitivity marker for MALT strains. Our data confirmed that H. pylori virulence factors are correlated with one another. If the involvement of H. pylori in MALT lymphoma is well established, the pathomechanism by which gastric lymphoma occurs remains to be identified.

Helicobacter species and liver diseases: association or causation?

The discovery of Helicobacter hepaticus as a causal agent of hepatitis and hepatocarcinoma in mice has stimulated interest in looking for Helicobacter spp in human liver samples. These bacteria could be a risk factor for the progression of liver disease to cirrhosis and hepatocellular carcinoma, especially among patients chronically infected with hepatitis C virus. We reviewed the studies done on this topic, and, with the exception of one, all studies reported an association between the presence of Helicobacter spp and liver disease. However, these data are weakened by the fact that Helicobacter spp DNA was detected but no bacteria could be grown, and by the difficulties in identifying the Helicobacter spp involved. More studies are therefore needed to confirm whether a causal association exits between the presence of Helicobacter spp in the liver and the development of cirrhosis and hepatocellular carcinoma.

Matrix-assisted laser-desorption/ionization BIOTYPER: experience in the routine of a University hospital

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) is positioned at the forefront of bacterial identification in the future. Its performance needed to be evaluated in a routine Bacteriology laboratory to determine its true benefits. A prospective study was carried out in the Bacteriology laboratory of the Pellegrin University Hospital in Bordeaux, France, from April to May 2009. Bacterial isolates from clinical samples were identified by conventional phenotypic bacteriological methods [Phoenix (Becton-Dickinson) or API strips (bioMérieux)] and in parallel with a mass spectrometer (Ultraflex III TOF/TOF and the biotyper database from Bruker Daltonics). In case of a discrepancy between these results at the genus level, a 16S rRNA and/or rpoB gene sequencing was performed. Of the 1013 bacteria tested, 837 (82.6%) were correctly identified at the species level by MALDI-TOF mass spectrometry (MS) without extraction and 189 after extraction, i.e. 986 (97.3%) were correctly identified at the species level by MALDI-TOF MS, vs. 945 (93.2%) by phenotypic methods. Indeed, the extraction step was necessary for only 15% of the isolates. These results were even better when considering the genus, reaching almost 99% with MALDI-TOF MS and 98% with phenotypic methods. The performance of MALDI-TOF MS is very attractive considering its efficiency and rapidity, and the technique constitutes a precious tool for bacteriological identification in a routine laboratory.

Human Bone Marrow-Derived Stem Cells Acquire Epithelial Characteristics through Fusion with Gastrointestinal Epithelial Cells

Bone marrow-derived mesenchymal stem cells (MSC) have the ability to differentiate into a variety of cell types and are a potential source for epithelial tissue repair. Several studies have demonstrated their ability to repopulate the gastrointestinal tract (GIT) in bone marrow transplanted patients or in animal models of gastrointestinal carcinogenesis where they were the source of epithelial cancers. However, mechanism of MSC epithelial differentiation still remains unclear and controversial with trans-differentiation or fusion events being evoked. This study aimed to investigate the ability of MSC to acquire epithelial characteristics in the particular context of the gastrointestinal epithelium and to evaluate the role of cell fusion in this process. In vitro coculture experiments were performed with three gastrointestinal epithelial cell lines and MSC originating from two patients. After an 8 day coculture, MSC expressed epithelial markers. Use of a semi-permeable insert did not reproduce this effect, suggesting importance of cell contacts. Tagged cells coculture or FISH on gender-mismatched cells revealed clearly that epithelial differentiation resulted from cellular fusion events, while expression of mesenchymal markers on fused cells decreased over time. In vivo cell xenograft in immunodeficient mice confirmed fusion of MSC with gastrointestinal epithelial cells and self-renewal abilities of these fused cells. In conclusion, our results indicate that fusion could be the predominant mechanism by which human MSC may acquire epithelial characteristics when in close contact with epithelial cells from gastrointestinal origin . These results could contribute to a better understanding of the cellular and molecular mechanisms allowing MSC engraftment into the GIT epithelium.

PCR-Restriction Fragment Length Polymorphism Analysis for Detection of Point Mutations Associated with Macrolide Resistance in Campylobacter spp.

ABSTRACT A 23S rRNA gene fragment in domain V was sequenced from 30 clinical isolates of Campylobacter spp., including 22 resistant to macrolides. Two point mutations associated with erythromycin resistance were identified at positions 2074 and 2075 on the 23S rRNA gene (homologous to A2142C and A2143G mutations in Helicobacter pylori) in which an adenine residue is also replaced with a cytosine and a guanine residue, respectively. A combined PCR-restriction fragment length polymorphism technique was developed to detect these mutations by using the BsaI and BceAI enzymes.

New Pathogenicity Marker Found in the Plasticity Region of the Helicobacter pylori Genome

ABSTRACT Comparison of gastric carcinoma and gastritis isolates showed the presence of genes, probably carcinoma associated (JHP947 and JHP940), that are situated in a Helicobacter pylori genome region (45 kb in J99 and 68 kb in 26695) called the “plasticity region.” This region presents a great variability of DNA sequences. We investigated, by PCR, the presence of the JHP940 and JHP947 genes, as well as the presence of a third gene which seems to be associated with gastritis (HP986), on H. pylori strains isolated from 200 Brazilian patients, 79 of whom had gastric carcinomas and 53 of whom had duodenal ulcers, to confirm this association. Gastritis isolates (n = 68) were included as a control. We also evaluated if these genes were related to the virulence-associated cagA genotype. The present methodology did not permit definitive conclusions to be reached regarding the association between the JHP940 gene and gastric carcinoma or between the HP986 gene and gastritis. However, we showed that the JHP947 gene might be implicated in the development of both duodenal ulcer and gastric carcinoma. The presence of the JHP947 gene was associated with the cagA-positive genotype. The JHP947 gene is a novel virulence marker candidate of H. pylori.