Martine Garnier

Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori

ABSTRACT We developed a single-vessel multiplex real-time PCR assay that detects Helicobacter pylori infection and identified the four existing alleles of the 23S rRNA genes of H. pylori—the wild-type sequence and the three mutations conferring clarithromycin resistance—using allele-specific Scorpion primers directly on biopsy specimens. The Scorpion primers combine a primer and a probe in a single molecule and are able to distinguish single-nucleotide polymorphism. Fluorescent signals, produced when the probes are annealed, are read in four channels by a SmartCycler thermocycler. The assay was first applied successfully on 4 reference and 61 clinical strains. MICs of clarithromycin were determined by the Etest method. A perfect concordance was obtained between Etest and Scorpion PCR. Mixed populations were better detected by Scorpion PCR. We examined 259 biopsies from 229 patients by culture, PCR-restriction fragment length polymorphism (RFLP), and Scorpion PCR. One biopsy, positive for culture, exhibited inhibitors for both PCR-RFLP and Scorpion PCR. Twelve biopsies were positive for PCR-RFLP and Scorpion PCR but negative for culture with concordant determination of mutations in the 23S rRNA genes by the two PCR assays. Three biopsies were positive for Scorpion PCR only. Compared to culture, the sensitivity of Scorpion PCR was 98.3% and the specificity was 92.5%. The Scorpion PCR assay provides a highly accurate, rapid, and precise method for the detection and determination of mutations conferring clarithromycin resistance to H. pylori.

T2182C mutation is not associated with clarithromycin resistance in Helicobacter pylori.

In a recent article, Khan et al. described a new mutation in the 23S rRNA gene, T2182C, conferring clarithromycin resistance to Helicobacter pylori (1). An A-to-G mutation at either position 2142 or position 2143 and an A-to-C mutation at position 2142 in the 23S rRNA gene have been shown to confer resistance to clarithromycin in H. pylori (5). In 2000, we isolated a clarithromycin-resistant (Clar) strain from a French patient (MIC > 256 μg/ml). Sequencing the 23S rRNA gene revealed a single T2182C mutation. To confirm the role of the T2182C mutation, we used the PCR fragment containing this mutation to transform a susceptible receptor strain. We obtained Clar transformants. These results were accepted for oral presentation at the 11th International Workshop on Campylobacter, Helicobacter and Related Organisms held in 2001 (3), but a final checking of the MIC determined by E-test revealed a mixed set of colonies. We observed small colonies in the susceptible area of the plate and big colonies in the resistant area. Subcultivation of isolated colonies provided two different clones. A Clas clone (MIC, 0.016 μg/ml) harbors the T2182C mutation and a wild-type sequence at positions 2142 and 2143. A Clar clone (MIC, 256 μg/ml) harbors both the T2182C mutation and an A2143G mutation. Randomly amplified polymorphic DNA patterns from the Clas and Clar isolates demonstrated that they are genetically identical. Coming back to the frozen gastric biopsy sample, we isolated 53 single colonies of H. pylori. Fifty-two colonies were from the Clas clone, and only one was from the Clar clone. It is possible that Khan et al. were misled, like we were, by a mixed population of a Clas clone and a Clar clone. It is surprising that they mention in their article the use of “pure culture from a single colony [for] further study” (1). T2182C is not a new mutation, and it has been already described in previous reports (2, 4, 6). In 1998, Wang et al. (6) first reported a Clar strain with A2143G and T2182C mutations. In vitro site-directed mutagenesis experiments suggested that this additional mutation is not associated with clarithromycin resistance (6). In 1999, Matsuoka et al. (4) reported the T2182C mutation in both sensitive and resistant colonies. In 2002, Kim et al. (2) described the same T2182C mutation for four Clar isolates, but they did not indicate that they worked on single-colony isolates. Attention must be paid to experiments used as evidence that a mutation is responsible for resistance in H. pylori. Experiments have to be realized on several isolated colonies, since a mixture of strains can lead to a false determination of sequence. Transformation with PCR fragments could lead to resistant clones growing on antibiotic plates, since the error rate of Taq polymerase spontaneously generates random mutations containing the well-known A2142G or A2143G mutations (5). The existence of a Clas isolate (20-222S) harboring a T2182C mutation in the 23S rRNA genes led to the conclusion that the T2182C mutation is not associated with clarithromycin resistance in H. pylori. The susceptible clone 20-222S is available on request.

Distribution of Spontaneous gyrA Mutations in 97 Fluoroquinolone-Resistant Helicobacter pylori Isolates Collected in France

ABSTRACT We determined the prevalence of gyrA mutations conferring fluoroquinolone resistance in 97 Helicobacter pylori isolates collected in France from 2007 to 2010. Ninety-four harbored one or two mutations already found in the quinolone resistance determining region (QRDR) of gyrA (for T87I, n = 23; for N87K, n = 32; for D91N, n = 30; for D91G, n = 7; for D91Y, n = 6), 2 harbored a mutation never previously described (D91H and A88P), and one strain was resistant (ciprofloxacin MIC of 8 mg/liter) without a detected mutation conferring this resistance in gyrA or gyrB genes.

Chemokines and Antimicrobial Peptides Have a cag-Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

ABSTRACT Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. The cag pathogenicity island (cag PAI) of H. pylori allows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response to H. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori B128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models with H. pylori B128ΔcagM, a cag PAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells with H. pylori, inflammatory-mediator production was largely due to cag PAI substrate-independent virulence factors. Thus, H. pylori cag PAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation during H. pylori infection.

Multiple and mixed Helicobacter pylori infections: Comparison of two epidemiological situations in Tunisia and France.

Individuals can be infected by either a single or multiple strains of Helicobacter pylori. Multiple infection with genetically different isolates and particularly mixed infection with both antibiotic-susceptible and resistant isolates are difficult to detect and should impact the effectiveness of eradication treatment. It is largely assumed that multiple infections are more frequent in developing countries but an actual comparison developing/developed using a single methodology has never been reported. To compare the prevalence of multiple and mixed H. pylori infection in Tunisia and France, we conducted a prospective study including 42 H. pylori-culture positive infected patients (21 Tunisian and 21 French) never previously treated for H. pylori infection. One gastric biopsy was collected from antrum. Three to eleven (mean = 9) colonies were isolated from each biopsy. A total of 375 different isolates were genotyped using RAPD fingerprinting and antimicrobial susceptibility testing was performed on amoxicillin, clarithromycin, ciprofloxacin, rifampicin, tetracycline and metronidazole with E-tests. Multiple infection was defined by different RAPD fingerprintings among the different isolates from a single patient. Mixed infection was defined by different resistance profiles among the different isolates from a single patient. Multiple H. pylori infection is more prevalent in Tunisia than in France. It occurred in ten (48%) Tunisian patients and in one (5%) French patient (p < 0.001). Mixed infection is common (24%), it occurred in 4 (19%) Tunisian patients and in 6 (29%) French patients (p = 0.46) and was mainly (8/10) due to genetically related clones in single infection.

Inflammatory signaling pathways induced by Helicobacter pylori in primary human gastric epithelial cells

Inflammatory signaling pathways induced by Helicobacter pylori remain unclear, having been studied mostly on cell-line models derived from gastric adenocarcinoma with potentially altered signaling pathways and nonfunctional receptors. Here, H. pylori-induced signaling pathways were investigated in primary human gastric epithelial cells. Inflammatory response was analyzed on chemokine mRNA expression and production after infection of gastric epithelial cells by H. pylori strains, B128 and B128ΔcagM, a cag type IV secretion system defective strain. Signaling pathway involvement was investigated using inhibitors of epidermal growth factor receptor (EGFR), MAPK, JAK and blocking Abs against TLR2 and TLR4. Inhibitors of EGFR, MAPK and JAK significantly reduced the chemokine mRNA expression and production induced by both H. pylori strains at 3 h and 24 h post-infection. JNK inhibitor reduced chemokine production at 24 h post-infection. Blocking Abs against TLR2 but not TLR4 showed significant reduction of chemokine secretion. Using primary culture of human gastric epithelial cells, our data suggest that H. pylori can be recognized by TLR2, leading to chemokine induction, and that EGFR, MAPK and the JAK/STAT signaling pathways play a key role in the H. pylori-induced CXCL1, CXCL5 and CXCL8 response in a cag pathogenicity island-independent manner.

In vitro culture and phenotypic and molecular characterization of gastric stem cells from human stomach.

Human gastric mucosa shows continuous self‐renewal via differentiation from stem cells that remain poorly characterized.

Pseudomonas aeruginosa flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis

ABSTRACT Pseudomonas aeruginosa, an opportunistic pathogen involved in skin and lung diseases, possesses numerous virulence factors, including type 2 and 3 secretion systems (T2SS and T3SS) and its flagellum, whose functions remain poorly known during cutaneous infection. Using isogenic mutants deleted from genes encoding each or all of these three virulence factors, we investigated their role in induction of inflammatory response and in tissue invasiveness in human primary keratinocytes and reconstructed epidermis. Our results showed that flagellum, but not T2SS and T3SS, is involved in induction of a large panel of cytokine, chemokine, and antimicrobial peptide (AMP) mRNA in the infected keratinocytes. Chemokine secretion and AMP tissular production were also dependent on the presence of the bacterial flagellum. This pro-inflammatory effect was significantly reduced in keratinocytes infected in presence of anti-toll-like receptor 5 (TLR5) neutralizing antibody. Bacterial invasion of human epidermis and persistence in a mouse model of sub-cutaneous infection were dependent on the P. aeruginosa flagellum. We demonstrated that flagellum constitutes the main virulence factor of P. aeruginosa involved not only in early induction of the epidermis inflammatory response but also in bacterial invasion and cutaneous persistence. P. aeruginosa is mainly sensed by TLR5 during the early innate immune response of human primary keratinocytes.