Jacques Tankovic

Single and Double Mutations in gyrA but Not in gyrB Are Associated with Low- and High-Level Fluoroquinolone Resistance in Helicobacter pylori

ABSTRACT In one French hospital the rate of resistance to ciprofloxacin in Helicobacter pylori was 3.3% (2 of 60 strains) in 1999. The six resistant clinical strains (four from 1996 and two from 1999) and three ciprofloxacin-selected single-step mutants studied carried one gyrA mutation but none in gyrB. Clinafloxacin and garenoxacin were the most active fluoroquinolones against these mutants. Occurrence of a second gyrA mutation was associated with high MICs of all fluoroquinolones tested.

Reduced activation of inflammatory responses in host cells by mouse‐adapted Helicobacter pylori isolates

Helicobacter pylori strains that harbour the Cag pathogenicity island (Cag PAI) induce interleukin (IL)‐8 secretion in gastric epithelial cells, via the activation of NF‐κB, and are associated with severe inflammation in humans. To investigate the influence of Cag PAI‐mediated inflammatory responses on H. pylori adaptation to mice, a selection of H. pylori clinical isolates (n= 12) was cag PAI genotyped and tested in co‐culture assays with AGS gastric epithelial cells, and in mouse colonization studies. Six isolates were shown to harbour a complete cag PAI and to induce NF‐κB activation and IL‐8 secretion in AGS cells. Of the eight isolates that spontaneously colonized mice, six had a cag PAI– genotype and did not induce pro‐inflammatory responses in these cells. Mouse‐to‐mouse passage of the two cag PAI+‐colonizing strains yielded host‐adapted variants that infected mice with bacterial loads 100‐fold higher than those of the respective parental strains (P= 0.001). These mouse‐adapted variants were affected in their capacity to induce pro‐inflammatory responses in host cells, yet no changes in cag PAI gene content were detected between the strains by DNA microarray analysis. This work provides evidence for in vivo selection of H. pylori bacteria with a reduced capacity to induce inflammatory responses and suggests that such bacteria are better adapted to colonize mice.

gyrA and gyrB Mutations Are Implicated in Cross-Resistance to Ciprofloxacin and Moxifloxacin in Clostridium difficile

ABSTRACT A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n = 100) and 1997 (n = 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 μg of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp71→Val in one, Thr82→Ile in six, and Ala118→Thr in one) or in gyrB (six mutations, amino acid changes Asp426→Asn in five and Arg447→Leu in one). These changes are similar to those already described in other species except for Asp71→Val, which is novel, and Ala118→Thr, which is exceptional. Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 μg/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.

Single or double mutational alterations of gyrA associated with fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli.

We looked for the presence of gyrA mutations in seven fluoroquinolone-resistant French clinical isolates of Campylobacter jejuni and Campylobacter coli. Three of the five isolates of C. jejuni and the two isolates of C. coli had high-level resistance to nalidixic acid (MICs 128-256 microg/ml) and ciprofloxacin (MICs 32 microg/ml). A gyrA mutation was found in all these isolates leading to the following substitutions: Thr86-Ile in four cases and Asp90-Tyr for one C. coli strain. One isolate had high-level resistance to nalidixic acid (MIC 64 microg/ml) but low-level resistance to ciprofloxacin (MIC 2 microg/ml) and also carried a gyrA mutation leading to a Thr86-Ala substitution. The last isolate of C. jejuni studied displayed an atypical resistance phenotype: It was resistant to high levels of ciprofloxacin (MIC 64 microg/ml) but remained fully susceptible to nalidixic acid (MIC 2 microg/ml). This phenotype was not explained by the presence of peculiar mutations in gyrA or gyrB. It carried a gyrA mutation leading to a Thr86-Ile substitution and was devoid of gyrB mutation. Despite numerous attempts with various degenerate oligonucleotide primers deduced from conserved regions of known parC genes, we were unable to amplify a corresponding sequence in C. jejuni or C. coli. First-step and second-step in vitro mutants, derived from reference strain C. coli ATCC 33559 with ciprofloxacin or moxifloxacin as selecting agents, were found to carry one and two mutations in gyrA, respectively. In contrast with the results obtained with clinical isolates, a variety of gyrA mutations were obtained in vitro.

Fast and Accurate Quantitative Detection of Helicobacter pylori and Identification of Clarithromycin Resistance Mutations in H. pylori Isolates from Gastric Biopsy Specimens by Real-Time PCR

ABSTRACT Rapid identification of patients infected with clarithromycin-resistant Helicobacter pylori without the need for culture can help to avoid useless prescriptions of clarithromycin. We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose. One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus. Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test). Susceptibility to clarithromycin was tested by E-test and PCR. The prevalence of infection was 33.7% (66 of 196 samples). The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively. The specificity of PCR was 94.6% (123 of 130 samples). The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample. For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR. Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant. The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR. Our PCR assay is accurate for fast detection of H. pylori as well as of clarithromycin resistance and is also able to objectively determine bacterial density.

Impairment of polymorphonuclear neutrophil functions precedes nosocomial infections in critically ill patients

Objective A postinjury immunodepression involving neutrophil functions has been described in critically ill patients. The aim of this prospective study was to search for a relationship between an impairment of neutrophil functions and the subsequent development of nosocomial infection. Design Twenty-one severely ill (simplified acute physiology score II >20 on admission), nonimmunosuppressed patients who were receiving no antibiotics active against methicillin-resistant Staphylococcus aureus and highly resistant Pseudomonas aeruginosa were included. Twelve healthy subjects constituted a control group. Measurements Neutrophil functions (phagocytosis and bactericidal activity toward S. aureus and P. aeruginosa in homologous plasma, reactive oxygen species secretion) were studied at day 4 ± 1 after admission, and occurrence of nosocomial infection was prospectively recorded over the following 5 days. Interleukin-10 concentration was assessed by enzyme-linked immunosorbent assay. Results are expressed as median (25th–75th percentiles). Main Results Six out of the 21 patients acquired a nosocomial infection during the 5 days after blood sampling (infected group). Compared with the patients who did not acquire nosocomial infection (noninfected group, n = 15), the neutrophils of the infected group demonstrated a higher percentage of intracellular bacterial survival (17% [2% to 67%] vs. infected: 62% [22% to 100%], p < .05), leading to an impairment of S. aureus killing in homologous plasma (killed bacteria: 4.93 log10 colony forming units/mL [4.24–5.29] vs. infected: 3.62 log10 colony forming units/mL [0.00–4.58], p < .05). Interleukin-10 plasma concentration was higher in infected patients (78 pg/mL [60–83]) compared with noninfected patients (22 pg/mL [14–58], p < .05). By contrast, P. aeruginosa killing was similar in patients whether or not they acquired a nosocomial infection. Conclusion A decrease in S. aureus killing capabilities of neutrophils can be evidenced within the days before occurrence of a nosocomial infection.

Frequent Association between Alteration of the rdxA Gene and Metronidazole Resistance in French and North African Isolates of Helicobacter pylori

ABSTRACT Mutations in the rdxA gene have been associated with the acquisition of resistance to metronidazole in Helicobacter pylori. This gene encodes an NADPH nitroreductase whose expression is necessary for intracellular activation of the drug. We wished to examine whether mutations in rdxA were present in resistant H. pylori isolates infecting either French or North African patients. We determined the complete nucleotide sequences of the rdxA genes from seven French and six North African patients infected with paired resistant and sensitive strains. Genotyping by random amplified polymorphic DNA analysis confirmed the close genetic relatedness of the susceptible and resistant isolates from individual biopsies. Eight French and five North African individual resistant strains were also studied. For the French strains, an alteration in rdxA most probably implicated in resistance was found in 10 cases (seven frameshift mutations, two missense mutations, and one deletion of 211 bp). One to three putative missense mutations were identified in four cases, and a missense mutation possibly not implicated in resistance was discovered in the last case. For the North African strains, an alteration inrdxA was found in eight cases (three frameshift mutations, three missense mutations, one deletion of 6 bp, and one insertion of a variant of IS605). Two strains contained putative missense mutations, and no change was observed in rdxA of the last strain. Thus, inactivation of the rdxA gene is frequently, but not always, associated with resistance to metronidazole in French and North African clinical isolates of H. pylori. In addition, a variety of alterations of rdxA are associated with the resistant phenotype.

Induction of nitric oxide synthase in vivo and cell injury in rat duodenal epithelium by a water soluble extract of Helicobacter pylori

1 Helicobacter pylori (Hp) infection, which involves the gastric antrum and duodenal mucosa, may be involved in peptic ulceration by stimulating the local release of cytoxic or pro‐inflammatory factors. 2 Nitric oxide (NO) is known to be cytotoxic at high concentration. The aim of the present study was therefore to investigate the ability of a water soluble extract of Hp to induce NO synthase in duodenal mucosa and epithelial cells following its administration in vivo in rats and determine its association with cell damage. 3 Administration of Hp water extract (4u2003mlu2003kg−1) led to the expression of the calcium‐independent inducible nitric oxide synthase (iNOS) after 4u2003h in the duodenum, determined as [14C]‐arginine conversion to citrulline. 4 This iNOS activity was not reduced by pretreatment with anti‐neutrophil serum (0.4u2003mlu2003kg−1, i.p., 3u2003h before challenge). However, dexamethasone pretreatment (1u2003mgu2003kg−1, i.v., 2u2003h before the extract), or administration of the NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME, 5u2003mgu2003kg−1, i.v., 2.5u2003h after the extract) reduced this activity. 5 Furthermore, iNOS was expressed in duodenal isolated epithelial cells 4u2003h after the i.v. challenge with the extract, at a time when the cellular viability was also reduced, as assessed by trypan blue exclusion. 6 Dexamethasone pretreatment, administration of L‐NAME, or pretreatment with polymyxin B (1u2003mgu2003kg−1, i.v.) which binds endotoxin, reduced both the iNOS activity and epithelial cell damage. 7 The induction of NO synthase by the Hp extract thus results in duodenal epithelial cell injury and such actions could play a role in pathogenesis of peptic ulcer disease.

An observational study of upper gastrointestinal bleeding in intensive care units: is Helicobacter pylori the culprit?

Objective:Upper gastrointestinal bleeding (UGIB) related to stress ulcers was formerly a fearsome complication of intensive care. The incidence of this event has decreased over the years. However, the morbidity, mortality, and causes of UGIB, particularly the etiologic role of Helicobacter pylori infection, are still controversial. Therefore, we prospectively assessed the incidence of UGIB in the intensive care unit (ICU) and evaluated the role of H. pylori infection. Design:A prospective observational study followed by a case-control study. Setting:Seven ICUs in the Paris area, five of them located in teaching hospitals. Patients:All patients admitted consecutively to seven ICUs during a 1-year period were monitored for signs of clinically relevant UGIB. Interventions:None. Measurements and Main Results:Only cases of endoscopically confirmed UGIB were analyzed. Patients whose hemorrhage originated from the stomach and/or duodenum were tested for H. pylori infection, by means of serology, histologic examination, and stool antigen detection. The possible association between H. pylori and UGIB was examined in a case-control study. Twenty-nine of the 4,341 patients admitted to the seven ICUs during the study period had clinically relevant, endoscopically confirmed UGIB (incidence, 0.67%; 95% confidence interval, 0.56%–0.77%). Ulcers were most frequently observed endoscopically. Patients who bled had a higher Simplified Acute Physiology Score (SAPS II) at admission (mean ± sd, 47 ± 14 vs. 36 ± 28; p < .001). Despite a higher in-ICU mortality rate among patients who bled (73% vs. 16%; p < .001), death was never due to bleeding. H. pylori infection was more frequent in patients who bled than in matched controls (36% vs. 16%; p = .04). Conclusions:Clinically relevant, endoscopically confirmed UGIB is a rare event in the ICU setting and tends to occur in severely ill patients. H. pylori infection is more frequent in patients with gastroduodenal hemorrhage than in nonbleeding patients.

Revisited distribution of nonfermenting Gram-negative bacilli clinical isolates

Nonfermenting Gram-negative bacilli (NF-GNB) are ubiquitous environmental opportunistic bacteria frequently misidentified by conventional phenotypic methods. The aim of this study was to determine the distribution of NF-GNB species by 16xa0S rRNA gene sequencing (used as reference method) and to compare performances of biochemical tests and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). From nine French hospitals, 188 NF-GNB isolates (except P. aeruginosa and A. baumannii) were prospectively collected from 187 clinical samples between December 2008 and May 2009. By using the genotypic approach, 173 (92%) and 188 (100%) isolates were identified to the species and genus level, respectively. They covered 35 species and 20 genera, with a predominance of Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Pseudomonas putida group bacteria. Of the 173 species-level identified strains, concordant identification to the species-level was obtained for 75.1%, 83% and 88.9% of isolates with API 20 NE strip, the VITEK-2 (ID-GN card) system and MALDI-TOF-MS, respectively. By excluding S. maltophilia isolates accurately identified by the three methods, genus-level identification was much higher for MALDI-TOF-MS (92.9%), compared with API 20 NE and VITEK-2 (76.2% and 80.8%, respectively). In conclusion, MALDI-TOF-MS represents a rapid, inexpensive, and accurate tool for routine identification of NF-GNB in human clinical samples.