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PCR detection of Helicobacter pylori infection in gastric biopsies allows the detection of this bacterium and the mutations associated with macrolide resistance. The aim of this study was to evaluate the performance of RIDA®GENE H. pylori PCR (r-Biopharm) on the ELITe InGenius System (Elitech). Two hundred gastric biopsies were obtained. These biopsies were ground in nutrient broth. Two hundred microliters of this suspension was treated with proteinase K, and then, 200 µL was transferred to an ELITe InGenius sample tube and tested using RIDA®GENE H. pylori PCR reagents. In-house H. pylori PCR was used as a reference. The sensitivity of RIDA®GENE H. pylori PCR with ELITe InGenius was 100%, the specificity was 98% (95% confidence interval (CI), 95.3–100%), the PPV was 98% (95% CI, 95.3–100%), and the NPV was 100% for the detection of H. pylori. All of these parameters were 100% for the categorization of macrolide resistance. The adaptation of RIDA®GENE H. pylori PCR reagents on the ELITe InGenius System was successful. This PCR is easy to use on this system.

PCR detection of Helicobacter pylori infection in gastric biopsies allows the detection of this bacterium and the mutations associated with macrolide resistance. The aim of this study was to evaluate the performance of RIDA®GENE H. pylori PCR (r-Biopharm) on a BD MAX™ System (Becton Dickinson). Two hundred ten gastric biopsies obtained were included. These biopsies were ground in nutrient broth. Two hundred microliters of this suspension was treated with proteinase K; 200 µL was transferred to a BD MAX™ sample tube then tested using RIDA®GENE H. pylori PCR reagents. In-house H. pylori PCR was used as a reference. The sensitivity of RIDA®GENE H. pylori PCR with BD MAX™ was 100%, the specificity was 99.08% (95% confidence interval (CI), 97.21-100%), the PPV was 99.02% (95% CI, 97.09-100%), and the NPV was 100% for the detection of H. pylori. The sensitivity was 97.14% (95% CI, 93.87-100%), the specificity was 100%, the PPV was 100%, and the NPV was 98.48% (95% CI, 96.08-100%) for categorization of macrolides resistance. The adaptation of RIDA®GENE H. pylori PCR on the BD MAX™ System is of considerable interest for microbiologists who seek to establish this assay in their laboratories.

BACKGROUND AND OBJECTIVES The present study describes the successful adaptation of an in-house Polymerase Chain Reaction (PCR) for Helicobacter pylori detection coupled with the main mutations associated with resistance to clarithromycin in ready-to-use PCR microwell strips. MATERIALS AND METHODS These microwell strips can be used on LightCycler® 480, and are delivered with nine microliters of the reaction mixture dispensed into 8-well microwell strips. An extraction control PCR targeting the β-globin household gene is amplified in the same run as H pylori detection. RESULTS AND CONCLUSION These microwell strips can be stored at -20°C for 1 year and left at room temperature and in the light for up to 4 h with no impact on the PCR results. Microwell strips can also undergo a thaw and refreeze cycle without impacting the PCR results. These PCR microwell strips are available for purchase from Eurogentec.

Antimicrobial resistance in campylobacters has been described worldwide. The emergence of multiresistant isolates, particularly among Campylobacter coli isolates, is concerning. New resistance mechanisms appear frequently, and DNA-sequence-based methods such as whole-genome sequencing (WGS) have become useful tools to monitor their emergence. The genomes of 51 multiresistant French Campylobacter sp. clinical strains from 2018 to 2019 were analyzed to identify associated resistance mechanisms. Analyses of erythromycin-resistant strains revealed 23S rRNA mutations among most of them and two different methyltransferases in 4 strains: Erm(B) and a novel methyltransferase, named Erm(N) here. The erm(B) gene was found in multidrug-resistant genomic islands, whereas erm(N) was inserted within CRISPR arrays of the CRISPR-cas9 operon. Moreover, using PCR screening in erythromycin-resistant strains from our collection, we show that erm(N) was already present in 3 French clinical strains 2 years before its first report in 2018 in Quebec, Canada. Bacterial transformations confirmed that the insertion of erm(N) into a CRISPR-cas9 operon can confer macrolide resistance. Campylobacter species are easily able to adapt to their environment and acquire new resistance mechanisms, and the emergence of methyltransferases in campylobacters in France is a matter of concern in the coming years.

The diagnosis of Helicobacter pylori infection can be made by using noninvasive tests. The detection of bacterial antigens in stool samples is a technique proposed by some suppliers. The objective of this study was to evaluate retrospectively the performances of the commercially available RIDA®QUICK Helicobacter and RIDASCREEN® Helicobacter (R-Biopharm) kits in detecting H. pylori antigens in stool samples. A collection of 132 stools was used in this study: 94 stools obtained from H. pylori-negative patients and 38 stools from H. pylori-positive patients. The performances (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV)) were evaluated for the RIDA®QUICK Helicobacter and RIDASCREEN® Helicobacter kits in comparison with real-time PCR results performed on gastric biopsies as well as culture. Discordant results, with respect to H. pylori status, were checked on the same day as the test by repeating the procedure. All of the readings concerning the RIDA®QUICK Helicobacter tests were concordant between 3 users, i.e., 94/94 negative tests and 34/38 positive tests. RIDASCREEN® Helicobacter tests were negative for all 94 H. pylori-negative samples and positive for 35/38 positive stools. Reading of the RIDA®QUICK Helicobacter tests was not a problem in routine practice. The RIDA®QUICK Helicobacter and RIDASCREEN® Helicobacter kits show good performances and can be included in the armamentarium of diagnostic tests for H. pylori infection.

The molecular mechanism of Helicobacter pylori resistance to tetracycline involves mutations in the primary binding site of the ribosome. A resistance or reduced susceptibility to tetracycline could be the result of single, double or triple mutations in the 16S rRNA gene of H. pylori . We investigated if the genotype was correlated to tetracycline resistance as determined phenotypically in vitro for 96 H . pylori isolates in the gastroesophageal mucosa of Venezuelan individual hosts. E-test for antimicrobial susceptibility test and real-time PCR for the detection of 16S rRNA gene mutations were performed in 96 H . pylori isolates (48 obtained from antrum, and 48 from oesophagus) from eight dyspeptic patients. In the gastric mucosa, 38 isolates were identified sensitive and 10 resistant to tetracycline by E-test, whereas 44 sensitive and 4 resistant isolates were found in the oesophagus. Real-time PCR detection of the 16S rRNA gene exhibited mutants with a single base-pair substitution (AGA926 GGA) in six antrum isolates and seven oesophagus isolates, whereas only three harboured a low level of tetracycline resistance in vitro. Our results indicate that real-time PCR detection of 16S rRNA is a reliable method to classify among tetracycline-resistant genotypes and useful in patients who have experienced a first-line treatment failure with triple therapy.

Background The prevalence of Helicobacter pylori infection was never assessed in Armenia, nor was the prevalence of H. pylori resistance against the main antibiotics concerned, despite the fact that these data are fundamental to establish evidence-based recommendations for management of this infection. We aimed to fill this gap by assessing prevalence of H. pylori among adult population in Armenia and resistance of H. pylori strains to clarithromycin and levofloxacin. Methods Helicobacter pylori seroprevalence was determined in 217 asymptomatic adult subjects submitted to a health checkup using an ELISA. Molecular methods were used to detect H. pylori in gastric biopsies from 91 adult dyspeptic patients [55 (60.4%) were positive] as well as the mutations associated with clarithromycin resistance by real-time PCR and with levofloxacin by sequencing the gyrA QRDR. Results Helicobacter pylori seropositivity was found to be 41.5% globally and increased with age from 13.6% (age 18–25 years) to 83.3% (age > 65 years). Only two cases were found with a A2142/43G mutation causing clarithromycin resistance, and 6 cases showed mutations associated with levofloxacin resistance. Conclusions Helicobacter pylori infection is estimated to be about 42% among adults in Armenia and the low clarithromycin resistance allows the use of the standard triple therapy as a first line therapy.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis worldwide. Mainly isolated from stool samples, C. jejuni can also become invasive. C. jejuni belongs to the commensal microbiota of a number of hosts, and infection by this bacterium can sometimes be traced back to exposure to a specific source. Here we genome sequenced 200 clinical isolates (2010–2016) and analyzed them with 701 isolate genomes from human infection, chicken, ruminants and the environment to examine the relative contribution of different reservoirs to non-invasive and invasive infection in France. Host-segregating genetic markers that can discriminate C. jejuni source were used with STRUCTURE software to probabilistically attribute the source of clinical strains. A self-attribution correction step, based upon the accuracy of source apportionment within each potential reservoir, improved attribution accuracy of clinical strains and suggested an important role for ruminant reservoirs in non-invasive infection and a potentially increased contribution of chicken as a source of invasive isolates. Structured sampling of Campylobacter in the clinic and from potential reservoirs provided evidence for variation in the contribution of different infection sources over time and an important role for non-poultry reservoirs in France. This provides a basis for ongoing genomic epidemiology surveillance and targeted interventions.

Introduction: CagA and VacA are the most important and well-studied virulence factors found in Helicobacter pylori. The aim of this work was to identify genes corresponding to H. pylori pathogenicity factors in Brazzaville, Congo. Material & Methods: A cross-sectional study was carried out from October 2013 to December 2016. Biopsy specimens were obtained from patients scheduled for upper gastrointestinal endoscopy in Brazzaville, Congo and were sent to the French National Reference Center for Campylobacters and Helicobacters in Bordeaux, France. H. pylori detection was conducted by real-time PCR using a fuorescence resonance energy transfer-melting curve analysis protocol. The identification of the genes encoding pathogenicity factors was carried out by conventional PCR using the appropriate primers for determination of CagA phosphorylation motifs 1, 2, 3; and vacAs, I and m regions: vacAi1, vacAi2, vacAs1a, vacAs1b. Results: A high prevalence of H. pylori infection was reported (108/143; 75.5%). In 92.2% (n = 71/77), the presence of P1, P2 and P3 CagA phosphorylation motifs was noted. Concerning vacA, vacAs1m1 was observed in 82% of the strains (n = 59/72). vacAi1 was present in all strains (n = 76). With regard to the distribution according to the vacAs1 subtype, the majority of the strains (59/71; 83%) were vacAs1b positive, as compared to vacAs1c (17/34, 33%). The vacAs1a gene was absent in all of these patients. Conclusion: The presence of genes associated with severe gastric diseases indicates the importance of H. pylori eradication in the prevention of these diseases in Congo.

Campylobacter spp. enteritis is the most frequent bacterial enteritis in both adults and children and is sometimes a source of severe complications. Its diagnosis by culture suffers from a lack of sensitivity and delays the result, preventing an early initiation of optimal antibiotic therapy in some cases. Our aim was to test a new rapid immuno-enzymatic method for Campylobacter spp. diagnosis in comparison to a composite reference standard (CRS). Stool samples from the French National Reference Center for Campylobacter and Helicobacter were tested with the CAMPYLOBACTER QUIK CHEK™ (Abbott). The CRS used to consider a case positive for Campylobacter spp. was a positive culture and, in case of a negative culture, a positive result obtained with both an ELISA and a molecular test. One hundred and eight stools were included: 53 were positive according to the CRS. If performed alone, culture would have missed 5 cases which the CAMPYLOBACTER QUIK CHEK™ detected. Finally, the CAMPYLOBACTER QUIK CHEK™ showed a sensitivity of 96.2% and a specificity of 94.5% and is relevant for clinical practice. Given the characteristics of the new method, it can be used as a screening method for Campylobacter spp. detection.

International audience; Background and objectives: Surveillance of Helicobacter pylori resistance to antibiotics was carried out in France in 2014, 2016, and 2018. We report here the results of the 2018 survey as well as the evolution over the 5-year period.Materials and methods: In this observational study, gastric biopsies were obtained by 62 gastroenterologists randomly selected in 5 regions of France and sent to a central laboratory where culture, antimicrobial susceptibility testing, and a real-time PCR were performed in order to detect H pylori and its mutations associated with clarithromycin resistance.Results and conclusion: During the year 2018, 951 patients were included: 55.3% women, mean age: 52.4 years ± 15.7, 71.6% born in France. Among them, 359 patients were H pylori positive by both culture and real-time PCR, and 7 more by PCR only. There were 244 naive patients, 110 previously treated patients, and unknown for 5. Primary resistance to clarithromycin was 20.9% [16.3-26.4], to levofloxacin 17.6% [13.4-22.9], and to metronidazole 58.6% [52.3%-64.6%]. Secondary resistance for these antibiotics was 56.4%, 22.7%, and 87.3%, respectively. There was no resistance to amoxicillin and tetracycline and very low resistance to rifampicin (1.2%) in both naive and treated patients. Primary resistance to clarithromycin decreased from 22.2% to 20.3% between 2014 and 2016, and appears to be stable since then. This can be linked to a stable consumption of macrolides over the 3-year time period. Primary levofloxacin resistance was relatively stable while metronidazole resistance increased. Interestingly, in both naive and treated patients, amoxicillin and rifampicin resistance were rare.

Background The diagnosis of Helicobacter pylori infection can be made by PCR on gastric biopsies. The objective of this study was to evaluate retrospectively the performance of the Allplex™ H pylori and ClariR PCR Assay (Seegene). Material and methods A collection of 180 DNA samples extracted from gastric biopsies was used in this study: 90 DNAs from H pylori-negative patients and 90 from H pylori-positive patients. The Allplex™ H pylori and ClariR Assay was performed on a CFX96™ real-time PCR System and analyzed using the Seegene Viewer software. The real-time PCR used as the reference was our in-house H pylori PCR, and discrepant results were tested by the Amplidiag® H pylori + ClariR PCR (Mobidiag). Results The performance of the Allplex™ H pylori and ClariR Assay showed 100% sensitivity, 97.6% specificity, 98% PPV, and 100% NPV. Regarding the detection of H pylori in the 90 expected negative samples, eight late amplifications were obtained (Ct > 39). Six of these eight samples were also positive using the Amplidiag® H pylori + ClariR kit and were therefore considered as true positives. For the two remaining cases, non-pathological evidence of H pylori infection was found. H pylori was detected in all 90 positive samples. Compared with our in-house H pylori PCR, all H pylori WT cases or mutated cases were correctly detected. Conclusions The Allplex™ H pylori and ClariR Assay showed an excellent performance and can be integrated into the armamentarium of diagnostic tests for H pylori infection. This kit has the advantage of differentiating the main mutations associated with macrolide resistance.

Arcobacter is considered an emergent foodborne enteropathogen. Despite the high prevalence of this genus in poultry, the occurrence of Arcobacter spp. contamination in Tunisia remains unclear. The objectives of this study were (i) to isolate Arcobacter species (A. butzleri and A. cryaerophilus) by the culture method from different species of raw poultry meat, (ii) to verify the isolates by multiplex PCR (m-PCR) assay and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and (iii) to determine the antibiotic resistance profiles of the isolates. A total of 250 poultry product samples (149 chicken and 101 turkey) were collected from various supermarkets in Sfax. The samples consisted of breasts, wings, legs, and neck skins. The overall isolation frequency of Arcobacter spp. was 10.4%. Arcobacter spp. were found in 13.42% of the chicken samples and in 5.49% of the turkey samples. All the acquired isolates were subject to detailed confirmation with subsequent species classification using m-PCR and MALDI-TOF MS. A. butzleri was found in 22 samples (84.61%) and A. cryaerophilus in 4 samples (15.38%). Thus, m-PCR and MALDI-TOF MS were able to detect A. butzleri significantly better than the conventional method (χ2 = 49.1 and P < 0.001). Arcobacter was isolated from poultry in every season, at contamination levels of 30.76, 23.07, 19.23, and 26.92% in summer, spring, autumn, and winter, respectively. The disk diffusion method was used to determine the susceptibility of Arcobacter isolates to six antimicrobial drugs. All A. butzleri isolates (n = 24) were significantly resistant to erythromycin (P = 0.0015), ampicillin (P = 0.001), and ciprofloxacin (P = 0.05). All tested A. cryaerophilus strains (n = 4) were susceptible to ampicillin, gentamicin, and amoxicillin-clavulanic acid. Multidrug resistance was observed in 83% of the Arcobacter spp. isolates. Our study detected Arcobacter spp. in Tunisian poultry; because of their multidrug resistance, these species may constitute a public health problem. HIGHLIGHTS

The aim of the present study was to propose epidemiologic cutoffs that could be used in routine practice to separate wild-type from non-wild-type Campylobacter fetus to ciprofloxacin. A total of 123 C. fetus isolates obtained from human samples were used for this purpose. Based on the determination of inhibition zone diameter, minimum inhibitory concentration, and sequencing of the quinolone resistance determining region in the gyraseA gene, for all tested isolates, the following cutoffs were proposed: ciprofloxacin-wild type if the inhibition zone diameter was ≥22 mm or the minimum inhibitory concentration was ≤0.5 mg/L.

Objectives In Algeria, there are limited data regarding the pattern of Helicobacter pylori primary antibiotic resistance. The aim of this study was to evaluate the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and to determine the molecular mechanisms involved in the resistance. Methods Two hundred and seventy Algerian adults who had never received H. pylori treatment were enrolled in this study. Human biopsies were obtained for culture and antimicrobial susceptibility testing was performed by Etest for clarithromycin, ciprofloxacin, tetracycline and rifampicin. Real-time fluorescence resonance energy transfer (FRET)-PCR was also performed in all cases to assess primary clarithromycin resistance and point mutations involved, real-time PCR was used to detect mutations involved in tetracycline primary resistance and sequencing of the QRDR of gyrA was performed to detect mutations involved in quinolone resistance. Results No resistance to rifampicin was detected. Resistance to clarithromycin and ciprofloxacin was found in 29.7% and 17.9%, respectively. Results of real-time FRET-PCR showed that A2143G was the most frequent point mutation, A2142C was not found and 42 patients (15.5%) were infected by both resistant and susceptible genotypes. Only two isolates were resistant to tetracycline and exhibited an A926G mutation. Four mutations were found to be responsible for resistance to ciprofloxacin [N87K (44.73%), D91N (23.68%), N87I (18.42%) and D91G (7.89%)]. Conclusions Local data regarding the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and the main genetic mutations involved in the resistance are necessary for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Algeria.

Thermophilic Campylobacter spp. are one of the primary causes of bacterial human diarrhea. The consumption of poultry meats, by-products, or both is suspected to be a major cause of human campylobacteriosis. The aims of this study were to determine the prevalence of thermophilic Campylobacter spp. in fresh poultry meat and poultry by-products by conventional culture methods and to confirm Campylobacter jejuni and Campylobacter coli isolates by using the multiplex PCR assay. Two hundred fifty fresh poultry samples were collected from a variety of supermarkets and slaughterhouses located in Sfax, Tunisia, including chicken (n =149) and turkey (n =101). The samples were analyzed using conventional microbiological examinations according to the 2006 International Organization for Standardization method (ISO 10272-1) for Campylobacter spp. Concurrently, a real-time PCR was used for identification of C. jejuni and C. coli . Of the 250 samples of poultry meat and poultry by-products, 25.6% (n = 64) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (26.8%) followed by turkey meat (23.7%). Among the different products, poultry breasts showed the highest contamination (36.6%) followed by poultry by-products (30%), poultry wings (28%) and poultry legs (26%) showed the lowest contamination, and no contamination was found on neck skin. Of the 64 thermophilic Campylobacter isolates, C. jejuni (59.7%) was the most frequently isolated species and 10.9% of the isolates were identified as C. coli . All of the 64 Campylobacter isolates identified by the conventional culture methods were further confirmed by PCR. The seasonal peak of Campylobacter spp. contamination was in the warm seasons (spring and summer). The study concluded that high proportions of poultry meat and poultry by-products marketed in Tunisia are contaminated by Campylobacter spp. Furthermore, to ensure food safety, poultry meats must be properly cooked before consuming.

Introduction La connaissance de la sensibilite aux antibiotiques qui evolue dans le temps est un element essentiel dans la prise en charge de l’infection a Helicobacter pylori. Le but de cette etude a ete de determiner la sensibilite aux principaux antibiotiques utilises pour cette infection en 2018 en France et de la comparer a celle observee dans deux etudes precedentes (2014 et 2016) menees selon le meme protocole. Materiels et methodes Il s’agit d’une etude observationnelle multicentrique. Des gastro-enterologues selectionnes au hasard dans 5 grandes regions metropolitaines ont recrute des patients adultes (maximum 1000) necessitant une fibroscopie. Les biopsies (antre et fundus) etaient transportees a 4 °C et traitees au CNR des Helicobacters. Apres broyage la culture etait realisee sur 2 milieux selectifs incubes a 37 ° C en atmosphere micro-aerobie durant un maximum de 10 jours. Les tests d’identification etaient pratiques sur les colonies suspectes ainsi qu’un antibiogramme par Etest (clarithromycine, levofloxacine, metronidazole, amoxicilline) ou disque (tetracycline, rifampicine). En parallele, un PCR en temps reel maison ciblant l’ADNr 23S etait effectuee pour detecter H. pylori et les eventuelles mutations associees a la clarithromycine. Resultats Finalement, 62 medecins ont ete recrutes et ont inclus 951 patients : hommes 44,7 %, âge moyen 52,4 ans et 79,5 % nes en Europe. H. pylori a ete detecte chez 359 par culture (37,7 %) et sept de plus par PCR. Parmi ces 366 cas positifs, 248 n’avaient jamais recu de traitement d’eradication. Le taux de resistance a la clarithromycine pour ces 248 etait de 20,9 %, proche de celui de 2016 (20,3 %) mais inferieur a celui de 2014 (22,2 %). En revanche, la resistance a la levofloxacine est en legere augmentation (15,4 %, 14,7 % et 17,6 %, respectivement en 2014, 2016 et 2018), ainsi que celle au metronidazole (45,9 %, 54,2 % et 58,6 %). Les cas de resistance a l’amoxicilline et a la rifampicine sont restes inferieurs ou proches de 1 % et aucune resistance a la tetracycline n’a ete observee, ni d’ailleurs chez les malades ayant eu un echec du Pylera®. Conclusion En conclusion, on observe une stabilisation de la resistance a la clarithromycine aux environs de 20 %, ce qui justifie la necessite de tester cet antibiotique avant de l’utiliser. La resistance a la levofloxacine est en legere augmentation et au metronidazole en nette augmentation. Une quasi absence de resistance est notee pour les autres antibiotiques. La strategie proposee par l’HAS de tester la sensibilite a la clarithromycine avant de prescrire une triple therapie contenant cet antibiotique est pleinement justifiee, d’autant qu’il existe des tests moleculaires disponibles dans ce but.

Campylobacter enteritis is the most frequent bacterial enteritis including in children. Its diagnosis suffers from the lack of sensitivity and delayed result of culture. Our aim was to test a new PCR-derived method for Campylobacter diagnosis in comparison to a composite reference. Patients presenting to the emergency ward of our hospital with enteric symptoms during the 2016 summer season were included. In addition to culture, an ELISA and an in-house real-time PCR were performed, as well as the new method (Orion GenRead Campylobacter) on all stool specimens. The composite reference used to consider a case positive for Campylobacter was either culture positive and in case of negative culture both the ELISA and real-time PCR positive. One hundred fifty patients were included, 64 being infants or children. There were 29 cases positive by the composite reference, with 19 of the 64 children (29.7%) and 10 of the 86 adults (11.6%). If performed alone, culture would have missed six cases. The Orion GenRead Campylobacter detected all the positives by the composite reference but also 12 cases negative by the composite reference (sensitivity 100%, specificity 90.1%). Given the characteristics of the new method, it can be used as a screening method for Campylobacter detection.

The aim of this study was to evaluate, using two independent polymerase chain reaction (PCR) formats, the results of Campylobacter detection by the BD MAXTM Enteric Bacterial Panel PCR (Becton Dickinson, Le Pont de Claix, France) in the absence of positive culture. A total of 77 samples found positive for Campylobacter on BD MAXTM but negative by culture were studied. Upon reception, one in-house real-time-PCR for Campylobacter sp. and a PCR with the RIDAGENE Bacterial Stool Panel (r-biopharm, Darmstadt, Germany) were performed. The data obtained using these two PCR formats were evaluated with respect to the cycle threshold (Ct) and fluorescence intensity values (FI) obtained on BD MAXTM. Ct and FI values were also obtained for 80 positive Campylobacter cases by culture. Among the 77 samples, 33 were positive with the two PCRs, and 37 remained negative. For the 33 double-positive PCRs samples, the Ct values obtained on BD MAXTM were lower than 30 in 93.9%, and FI > 2000 for 97% of cases. For the 37 double-negative PCRs samples, the Ct values obtained on BD MAXTM were 2000 for 40.5% of cases. Positive culture cases had Ct values < 30 in 96.2% and FI > 2000 in 98.8%. We showed that the Ct values obtained on BD MAXTM can help to interpret the results. Almost 96% of the Campylobacter sp. cases detected by culture or with the two reference PCRs positive showed a Ct value on BD MAXTM, meaning that stools detected as positive with BD MAXTM and having a Ct > 30 may be false positives.

Helicobacter pylori is a major human pathogen. Diagnosis of H. pylori infection and determination of its antibiotic susceptibility still mainly rely on culture and phenotypic drug susceptibility testing (DST) that is time-consuming and laborious. Whole genome sequencing (WGS) has recently emerged in medical microbiology as a diagnostic tool for reliable drug resistance prediction in bacterial pathogens. The aim of this study was to compare phenotypic DST results with the predictions based on the presence of genetic determinants identified in the H. pylori genome using WGS. Phenotypic resistance to clarithromycin, metronidazole, tetracycline, levofloxacin, and rifampicin was determined in 140 clinical H. pylori isolates by E-Test&reg, and the occurrence of certain single nucleotide polymorphisms (SNPs) in target genes was determined by WGS. Overall, there was a high congruence of &gt, 99% between phenotypic DST results for clarithromycin, levofloxacin, and rifampicin and SNPs identified in the 23S rRNA, gyrA, and rpoB gene. However, it was not possible to infer a resistance phenotype for metronidazole based on the occurrence of distinct SNPs in frxA and rdxA. All 140 H. pylori isolates analysed in this study were susceptible to tetracycline, which was in accordance with the absence of double or triple nucleotide substitutions in the 16S rRNA gene.

International audience; Helicobacter pylori infection is responsible for gastric carcinogenesis but host factors are also implicated. IQGAP1, a scaffolding protein of the adherens junctions interacting with E-cadherin, regulates cellular plasticity and proliferation. In mice, IQGAP1 deficiency leads to gastric hyperplasia. The aim of this study was to elucidate the consequences of IQGAP1 deletion on H. pylori-induced gastric carcinogenesis.Transgenic mice deleted for iqgap1 and WT littermates were infected with Helicobacter sp., and histopathological analyses of the gastric mucosa were performed. IQGAP1 and E-cadherin expression was evaluated in gastric tissues and in gastric epithelial cell lines in response to H. pylori infection. The consequences of IQGAP1 deletion on gastric epithelial cell behaviour and on the acquisition of cancer stem cell (CSC)-like properties were evaluated. After one year of infection, iqgap1+/- mice developed more preneoplastic lesions and up to 8 times more gastro-intestinal neoplasia (GIN) than WT littermates. H. pylori infection induced IQGAP1 and E-cadherin delocalization from cell-cell junctions. In vitro, knock-down of IQGAP1 favoured the acquisition of a mesenchymal phenotype and CSC-like properties induced by H. pylori infection.Our results indicate that alterations in IQGAP1 signalling promote the emergence of CSCs and gastric adenocarcinoma development in the context of an H. pylori infection.

Aim: Helicobacter pylori infection is a worldwide infection, its eradication rates with conventional therapies have fallen to unacceptable levels. In this context we were interested in metformin, to determine its effect on H. pylori growth. Materials & methods: Antimicrobial susceptibility tests and survival curves were performed in vitro and a H. pylori-infected mice model was used to determine metformin effect in vivo. Results: Helicobacter pylori survival and growth were decreased in presence of metformin. Furthermore, metformin-treated mice had significantly less bacteria in their stomach than the untreated mice. Conclusion: Our work is the first to demonstrate a direct antimicrobial effect of metformin on H. pylori, indicating that this molecule has not yet revealed its full potential.

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113490/ A (WGS) approach was conducted in order to identify the molecular determinants associated with antimicrobial resistance in 12 multidrug-resistant Campylobacter jejuni and Campylobacter coli isolates, with a focus on aminoglycoside resistance determinants. Two variants of a new aminoglycoside phosphotransferase gene [aph(2″)-Ii1 and aph(2″)-Ii2 ] putatively associated with gentamicin resistance were found. In addition, the following new genes were identified for the first time in Campylobacter: a lincosamide nucleotidyltransferase gene [lnu(G)], likely associated with lincomycin resistance, and two resistance enzyme genes (spw and apmA) similar to those found in Staphylococcus aureus, which may confer spectinomycin and gentamicin resistance, respectively. A C1192T mutation of the 16S rRNA gene that may be involved in spectinomycin resistance was also found in a C. coli isolate. Genes identified in the present study were located either on the bacterial chromosome or on plasmids that could be transferred naturally. Their role in aminoglycoside resistance remains to be supported by genetic studies. Regarding the other antimicrobial agents studied, i.e., ampicillin, ciprofloxacin, erythromycin, and tetracycline, a perfect correlation between antimicrobial phenotypes and genotypes was found. Overall, our data suggest that WGS analysis is a powerful tool for identifying resistance determinants in Campylobacter and can disclose the full genetic elements associated with resistance, including antimicrobial compounds not tested routinely in antimicrobial susceptibility testing. This work was supported by internal funding of the Ricardo Jorge National Institute of Health. WGS was performed at the Unidade de Tecnologia e Inovação (Departamento de Genética Humana, Instituto Nacional de Saúde Ricardo Jorge, Lisbon, Portugal). info:eu-repo/semantics/publishedVersion

Antibiotic resistance is a major cause of the increasing failures in the current eradication therapies against Helicobacter pylori. In this scenario, repurposing drugs could be a valuable strategy to fast-track novel antimicrobial agents. In the present study, we analyzed the inhibitory capability of 1,4-dihydropyridine (DHP) antihypertensive drugs on the essential function of the H. pylori response regulator HsrA and investigated both the in vitro antimicrobial activities and the in vivo efficacy of DHP treatments against H. pylori. Six different commercially available and highly prescribed DHP drugs&mdash, namely, Nifedipine, Nicardipine, Nisoldipine, Nimodipine, Nitrendipine, and Lercanidipine&mdash, noticeably inhibited the DNA binding activity of HsrA and exhibited potent bactericidal activities against both metronidazole- and clarithromycin-resistant strains of H. pylori, with minimal inhibitory concentration (MIC) values in the range of 4 to 32 mg/L. The dynamics of the decline in the bacterial counts at 2 &times, MIC appeared to be correlated with the lipophilicity of the drugs, suggesting different translocation efficiencies of DHPs across the bacterial membrane. Oral treatments with 100 mg/kg/day of marketed formulations of Nimodipine or Nitrendipine in combination with omeprazole significantly reduced the H. pylori gastric colonization in mice. The results presented here support a novel therapeutic solution for treatment of antibiotic-resistant H. pylori infections.

The aim of this study was to evaluate, using two independent polymerase chain reaction (PCR) formats, the results of Campylobacter detection by the BD MAXTM Enteric Bacterial Panel PCR (Becton Dickinson, Le Pont de Claix, France) in the absence of positive culture. A total of 77 samples found positive for Campylobacter on BD MAXTM but negative by culture were studied. Upon reception, one in-house real-time-PCR for Campylobacter sp. and a PCR with the RIDAGENE Bacterial Stool Panel (r-biopharm, Darmstadt, Germany) were performed. The data obtained using these two PCR formats were evaluated with respect to the cycle threshold (Ct) and fluorescence intensity values (FI) obtained on BD MAXTM. Ct and FI values were also obtained for 80 positive Campylobacter cases by culture. Among the 77 samples, 33 were positive with the two PCRs, and 37 remained negative. For the 33 double-positive PCRs samples, the Ct values obtained on BD MAXTM were lower than 30 in 93.9%, and FI &gt, 2000 for 97% of cases. For the 37 double-negative PCRs samples, the Ct values obtained on BD MAXTM were &lt, 30 in only 18.9%, however FI were &gt, 2000 for 40.5% of cases. Positive culture cases had Ct values &lt, 30 in 96.2% and FI &gt, 2000 in 98.8%. We showed that the Ct values obtained on BD MAXTM can help to interpret the results. Almost 96% of the Campylobacter sp. cases detected by culture or with the two reference PCRs positive showed a Ct value on BD MAXTM, meaning that stools detected as positive with BD MAXTM and having a Ct &gt, 30 may be false positives.

Background Knowledge of antimicrobial susceptibility, especially to macrolides, has become crucial for the management of Helicobacter pylori infection. Our aim was to evaluate two new PCR kits able to detect H. pylori in gastric biopsies as well as the mutations associated with macrolide resistance. Materials and methods Two hundred successive biopsies (received from gastroenterologists all over France) were used. The two new kits tested were Amplidiag H. pylori+ClariR from Mobidiag Espoo, Finland, and RIDA® GENE H. pylori from R-Biopharm, Darmstadt, Germany. Culture and a validated in-house real-time PCR were also performed, and in the case of a positive culture, Etest for clarithromycin was carried out. Discrepancies were solved by looking at the pathologic data. Results Culture was positive in 68 cases (34%), and with our in-house real-time PCR in these 68 cases plus 5 others (N = 73, 36%). All were also detected by the two new kits. In addition, RIDA® GENE H. pylori detected one more positive also detected by Amplidiag H. pylori+ClariR , and Amplidiag detected two other positives. Of these three additional cases, pathology confirmed the positivity for two. Only one case diagnosed by Amplidiag could be considered as a false positive. With regard to clarithromycin resistance, 22 cases were detected. The corresponding mutations (A2142/43G) were all identified with the three PCRs. Conclusions These two new kits which have an excellent sensitivity and specificity are convenient to use, adaptable to different thermocyclers, provide quick results, and deserve to be used in H. pylori diagnosis for a better choice of treatment regimen.

International audience; Primary antibody deficiency (PAD) is characterized by a defective immunoglobulin production and recurrent infections, mostly involving respiratory and gastrointestinal tracts. Chronic or recurrent diarrhea is reported in up to 23%. Campylobacter infection is a common cause of infectious diarrhea, reported in 1.2% to 7.5% of patients with common variable immunodefi-ciency (CVID), the most frequent PAD. The aim of this study was to describe Campylobacter infection in patients with PAD included in a large nationwide study and analyze factors associ-ated with susceptibility to this pathogen. The DEFI (DEFicit Immunitaire) study is an ongoing large cross-sectional French multicentric study of adults with PAD, with retrospective collection of clinical data. All patients with a history of bacteriologically documented Campylobacter infection were identified, and clinical data were collected for each episode. Factors associated with recurrent infection were assessed as oddsratio (OR) and 95% confidence interval (CI), calculated by means of simple regression analysis. In patients with available material, strains of each episode were characterized using molecular analysis and compared (Table E1, available in this article’s Online Repository at www.jaci-inpractice.org). A com-parison of immunodeficiency-related characteristics of patients with and without Campylobacter infection was performed in the homogeneous group of patients with CVID. The control group included patients with CVID from DEFI centers who confirmed that patients did not develop Campylobacter infection after enrollment (Figure E1, available in this article’s Online Repository at www.jaci-inpractice.org). After correction for multiple comparisons, P

Open Access; International audience; We isolated from aerobic and anaerobic blood culture bottles from a febrile patient, a Helicobacter-like Gram negative, rod-shaped bacterium that MALDI-TOF MS failed to identify. Blood agar cultures incubated in a microaerobic atmosphere revealed a motile Gram negative rod, which was oxidase, catalase, nitrate reductase, esterase, and alkaline phosphatase positive. It grew at 42 • C with no detectable urease activity. Antimicrobial susceptibility testing showed that the organism was susceptible to beta-lactams, gentamicin, erythromycin, and tetracycline but resistant to ciprofloxacin. Electronic microscopy analysis revealed a 3 × 0.5 µm curved rod bacterium harboring two sheathed amphitrichous flagella. Whole genome sequencing revealed a genome 1,708,265 base-pairs long with a GC content of 37.80% and a total of 1,697 coding sequences. The genomic analyses using the nucleotide sequences of the 16S rRNA gene, hsp60 and gyrB genes, as well as the GyrA protein sequence, and the results of Average Nucleotide Identity and in silico DNA-DNA hybridization suggest evidence for a novel Helicobacter species close to Helicobacter equorum and belonging to the group of enterohepatic Helicobacter species. As soon as the particular peptide mass fingerprint of this pathogen is added to the spectral databases, MALDI-TOF MS technology will improve its identification from clinical specimens, especially in case of " sterile infection ". We propose to associate the present strain with the Latin name of the place of isolation; Caesarodunum (Tours, France) and suggest " Helicobacter caesarodunensis " for further description of this new bacterium.

Antimicrobial susceptibility testing is needed to adapt Helicobacter pylori treatment to obtain the best results. Beside the standard phenotypic methods, molecular methods are increasingly used. The value of these molecular tests is that they are quick, independent of the transport conditions, easy to standardize, and commercial kits are available. In this article, these methods are reviewed, focusing on the determination of H pylori resistance to macrolides and fluoroquinolones, and mentioning also the methods used for tetracycline and rifampin.

Helicobacter pylori (H. pylori) infection triggers a sequence of gastric alterations starting with an inflammation of the gastric mucosa that, in some cases, evolves to gastric cancer. Efficient vaccination has not been achieved, thus it is essential to find alternative therapies, particularly in the nutritional field. The current study evaluated whether curcumin could attenuate inflammation of the gastric mucosa due to H. pylori infection. Twenty-eight C57BL/6 mice, were inoculated with the H. pylori SS1 strain, ten non-infected mice were used as controls. H. pylori infection in live mice was followed-up using a modified 13C-Urea Breath Test (13C-UBT) and quantitative real-time polymerase chain reaction (PCR). Histologically confirmed, gastritis was observed in 42% of infected non-treated mice at both 6 and 18 weeks post-infection. These mice showed an up-regulation of the expression of inflammatory cytokines and chemokines, as well as of toll-like receptors (TLRs) and MyD88, at both time points. Treatment with curcumin decreased the expression of all these mediators. No inflammation was observed by histology in this group. Curcumin treatment exerted a significant anti-inflammatory effect in H. pylori-infected mucosa, pointing to the promising role of a nutritional approach in the prevention of H. pylori induced deleterious inflammation while the eradication or prevention of colonization by effective vaccine is not available.

Background Helicobacter pylori infection is common in Algeria, but there are few data on the characterization of isolated strains. The aim of this study was to update data on the prevalence of H. pylori in patients submitted to endoscopy, antibiotic resistance, and phylogeography of H. pylori strains isolated in Algiers. Materials and Methods This is a prospective study carried out between November 2015 and August 2016. The culture of H. pylori was performed on antral and fundic gastric biopsies of adult patients from 3 hospitals. A real-time PCR using the fluorescence resonance energy transfer (FRET) principle for the detection of H. pylori followed by a melting curve analysis for the detection of mutations associated with resistance to clarithromycin was applied. Differentiation between antral and fundic isolates of the same patient was also determined by RAPD, and an MLST typing was performed for characterization of the phylogeographic group of H. pylori. Results By real-time PCR, the prevalence of H. pylori infection among the 147 patients included was 57%. Culture was positive in only 29% of the cases. Twenty-seven percent of patients had received H. pylori eradication treatment. The primary and secondary resistance rates to clarithromycin were 23% and 36%, respectively, and to metronidazole, 45% and 71%, respectively. Only one isolate was resistant to levofloxacin, and no resistance to amoxicillin, tetracycline, and rifampicin was detected. A double population was present in 14 patients. The MLST analysis classified the 42 H. pylori strains from 38 patients in 2 haplotypes: hpEurope (33) and hpNEAfrica (9). Conclusion The prevalence of H. pylori remains high in Algeria but appears to be decreasing in recent years. High resistance to clarithromycin requires increased monitoring of the evolution of antibiotic resistance and adaptation of eradication therapy.

A large survey of antimicrobial resistance of Helicobacter pylori was performed in France in 2014: 984 patients were enrolled by 75 gastroenterologists all over the country. Among the 783 patients who had never received eradication treatment before, 266 (33.9%) were H. pylori positive. The strains showed a high rate of clarithromycin resistance (22.2%), moderate rate of resistance to levofloxacin (15.4%) and high rate of resistance to metronidazole (45.9%). In all, 187 patients had received previous treatment, of which 115 were H. pylori positive with very high resistance to clarithromycin (73.9%) and metronidazole (78.3%). None of the patients receiving PYLERA (Bismuth salt-Tetracycline HCl-Metronidazole) proton-pump inhibitor developed resistance to tetracycline. A real-time PCR applied to gastric biopsy specimens detected all the cases that were positive by culture as well as 30 additional cases. A good correlation was found between the clarithromycin resistance detected by phenotypic methods and the associated mutations for clarithromycin resistance, which has continued to increase in the last decade but at a lower rate than previously observed.

Back ground Helicobacter pylori infection is involved in several gastroduodenal diseases which can be cured by antimicrobial treatment. The aim of this study was to determine the prevalence of H. pylori infection and its bacterial resistance to clarithromycin, fluoroquinolones, and tetracycline in Brazzaville, Congo, by using molecular methods. Material and methods A cross- sectional study was carried out between September 2013 and April 2014. Biopsy specimens were obtained from patients scheduled for an upper gastrointestinal endoscopy and were sent to the French National Reference Center for Campylobacters and Helicobacters where they were tested by molecular methods for detection of H. pylori and clarithromycin resistance by real-time PCR using a fluorescence resonance energy transfer-melting curve analysis (FRET-MCA) protocol, for detection of tetracycline resistance by real-time PCR on 16S rRNA genes (rrnA and rrnB), for detection of point mutations in the quinolone resistance-determining regions (QRDR) of H. pylori gyrA gene, associated with resistance to quinolones, by PCR and sequencing. Results This study showed a high H. pylori prevalence (89%), low rates of clarithromycin and tetracycline resistance (1.7% and 2.5%, respectively), and a high rate of quinolone resistance (50%). Conclusion Therefore, the use of standard clarithromycin-based triple therapy is still possible as an empiric first-line treatment as well as prescription of bismuth-based quadruple therapy, which includes tetracycline, but not a levofloxacin-based triple therapy because of the high rate of resistance to fluoroquinolones.

Diarrhea is a frequent complication after kidney transplantation, ascribed to adverse effects of the immunosuppressive therapy in case of negative microbiological examination of the stools. The aim of this study was to improve the microbiological diagnosis by implementing molecular tests. Fifty-four severe diarrhea events that occurred in 49 adult kidney transplant recipients from September 2010 to November 2011 were investigated. One or several enteric pathogens were detected in 13 (23%) stool samples using classical microbiological methods versus 39 (72%) for the seven commercially available multiplex PCR assays used retrospectively ( P = 0.006). Interestingly, molecular diagnosis identified 15 multiple infections compared to none using classical techniques. The primary pathogens detected were enteropathogenic Escherichia coli (EPEC) ( n = 15; 38%), Campylobacter spp. ( n = 15; 38%), and Norovirus ( n = 14; 36%). Specificities for Campylobacter and Norovirus infection diagnosis were 75 and 100%, respectively, by comparison to reference methods. Based on molecular findings, a cyclosporine-mycophenolate mofetil combination was identified as a risk factor for developing Norovirus -induced diarrhea. Norovirus infections were also responsible for higher weight loss than all the other causes of diarrhea. In samples from asymptomatic immunocompromised and immunocompetent patients, EPEC but not Norovirus and Campylobacter infections were detected at a frequency similar to that observed in symptomatic kidney transplant recipients. In conclusion, molecular tools significantly improved the detection of single and multiple enteric infections by comparison to classical techniques and could quickly become the key element in the management of severe acute diarrhea in transplant recipients.