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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 16 S 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

MICs of the new fluoroquinolone, moxifloxacin, and those of ciprofloxacin, ofloxacin and sparfloxacin for 19 genetically characterized fluoroquinolone-resistant strains were determined by the agar dilution method. The MICs of moxifloxacin for Escherichia isolates with one mutation in gyrA (corresponding to Ser83→Leu or Asp87→Gly substitution) were 0.25–0.5 mg/L, while those of ciprofloxacin, ofloxacin and sparfloxacin were 0.06–0.25, 1 and 0.12–0.5 mg/L, respectively. These values were four- to 16-fold higher than those of the same antibiotics for the wild-type strain, E. coli KL16. Similar results were observed with clinical isolates of Salmonella spp. harbouring one mutation in gyrA leading to the substitution of Ser83 by Phe or Tyr. In the presence of two mutations in the E. coli gyrA gene, the MICs of moxifloxacin ciprofloxacin, ofloxacin and sparfloxacin were 2, 0.5, 4 and 1 mg/L, respectively; these were 32 times higher than the MICs of these agents for E. coli KL16. The MICs of the four quinolones for triple mutants with two mutations in gyrA and one in parC were even higher, i.e. 8, 8, 16 and 8–16 mg/L, respectively. The MICs of moxifloxacin for Campylobacter coli and Campylobacter jejuni strains with a gyrA mutation leading to Thr86→Ile substitution ranged from 1 to 2 mg/L, while the MICs of ciprofloxacin, ofloxacin and sparfloxacin were 16–32 mg/L, 8–16 and 4–8 mg/L, respectively. For high-level ciprofloxacin-resistant (MICs of 32 mg/L) clinical isolates of Enterococcus faecalis with one substitution at position 83 in GyrA (E. coli coordinates), the MICs of moxifloxacin, ofloxacin and sparfloxacin were 8–16, ≥128 and 32 mg/L respectively. In conclusion, moxifloxacin and other fluoroquinolones exhibit cross-resistance against aerobic Gram-negative bacilli and enterococci. The in-vitro activity of moxifloxacin was greater than that of ofloxacin and slightly less than that of ciprofloxacin and sparfloxacin against Enterobacteriaceae, but greater than those of the three other compounds tested against Campylobacter spp and E. faecalis. [ABSTRACT FROM PUBLISHER]

L'importance d'une antibiothérapie précoce et adéquate pour les bactériémies sévères et/ou sur terrain fragilisé est capitale. Le PhenoTest (Accelerate) permet, à partir d'une hémoculture positive, d'obtenir l'identification en 2 h (17 germes cibles, hybridation FISH) et l'antibiogramme en 7 h (analyse cellulaire morphocinétique avec rendu d'une CMI). Nous avons évalué de façon observationnelle cette technologie pour les patients d'hématologie et de réanimation médicale de notre hôpital. De février à décembre 2019, 51 flacons d'hémocultures positifs provenant de 47 patients avec sepsis (51 épisodes indépendants, 29 de réanimation et 22 d'hématologie) ont été étudiés. Après coloration de Gram, les flacons ont été analysés par les techniques de routine (spectrométrie de masse sur colonies, diffusion en gélose) et par le PhenoTest. Les résultats et leur temps d'obtention ont été comparés. L'impact de la documentation rapide sur la prise en charge a été évalué. L'étude a porté sur : – 42 bacilles Gram négatif (BGN) : 33 entérobactéries, 6 pyocyaniques, 2 bactéroïdes , 1 Stenotrophomonas ; – 1 bacille Gram positif (BGP) de type Bacillus , pris à tort pour un BGN car trop décoloré ; – 7 cocci Gram positif (CGP) : 4 entérocoques, 2 SCN et 1 pneumocoque ; – 1 levure (C. parapsilosis). Identification par PhenoTest : – BGN : 33 identifications correctes à l'espèce (79 %), 3 au genre entérobactérie (7 %), 3 hors panel (Stenotrophomonas et bactéroïdes) (7 %), 3 échecs (3 entérobactéries) (7 %) ; – BGP : hors panel ; – CGP : 5 identifications correctes (71,4 %), 1 hors panel (Enterococcus raffinosus), 1 échec (pneumocoque) ; – levure : hors panel. Un antibiogramme était rendu par PhenoTest pour : – 29/39 (74,4 %) BGN de type entérobactérie (25/33) ou pyocyanique (4/6) ; – les 5 CGP identifiables par PhenoTest (3 E. faecium et 2 SCN). Pour les BGN, les taux de discordance des antibiogrammes étaient : 2,9 % d'erreurs mineures, 1,0 % d'erreurs majeures et 0,5 % d'erreurs très majeures. Les gains de temps moyens étaient 22 h pour l'identification et 14 h pour l'antibiogramme. L'analyse rétrospective de l'impact potentiel sur l'antibiothérapie est en cours. Le PhenoTest a permis d'identifier en 2 h à l'espèce (ou au genre entérobactérie pour 3 cas) 84 % des 49 hémocultures à BGN et CGP testées. Il a permis de rendre un antibiogramme en 7 h pour 69 % des CGP et BGN testés, pour 74 % des BGN de type entérobactérie ou pyocyanique. La concordance de l'antibiogramme avec la diffusion en gélose est excellente. Il permet donc une adaptation rapide de l'antibiothérapie probabiliste des bactériémies à CGP, entérobactérie et pyocyanique dans environ ¾ des cas. [ABSTRACT FROM AUTHOR]

Background: The optimal treatment regimen for infections caused by wild-type AmpC β-lactamase-producing Enterobacterales remains controversial. This study compared the outcomes of bloodstream infections (BSI) and pneumonia according to the type of definitive antibiotic therapy: third-generation cephalosporin (3GC), piperacillin ± tazobactam, cefepime or carbapenem., Methods: All cases of BSI and pneumonia caused by wild-type AmpC β-lactamase-producing Enterobacterales over 2 years in eight university hospitals were reviewed. Patients who received definitive therapy consisting of either a 3GC (3GC group), piperacillin ± tazobactam (piperacillin group), or cefepime or a carbapenem (reference group) were included in this study. The primary endpoint was 30-day all-cause mortality. The secondary endpoint was treatment failure due to infection by emerging AmpC-overproducing strains. Propensity-score-based models were used to balance confounding factors between groups., Results: In total, 575 patients were included in this study: 302 (52%) with pneumonia and 273 (48%) with BSI. Half (n=271, 47%) received cefepime or a carbapenem as definitive therapy, 120 (21%) received a 3GC, and 184 (32%) received piperacillin ± tazobactam. Compared with the reference group, 30-day mortality was similar in the 3GC [adjusted hazard ratio (aHR) 0.86, 95% confidence interval (CI) 0.57-1.31)] and piperacillin (aHR 1.20, 95% CI 0.86-1.66) groups. The likelihood of treatment failure was higher in the 3GC (aHR 6.81, 95% CI 3.76-12.4) and piperacillin (aHR 3.13, 95% CI 1.69-5.80) groups. The results were similar when stratifying the analysis on pneumonia or BSI., Conclusion: Treatment of included BSI or pneumonia caused by wild-type AmpC β-lactamase-producing Enterobacterales with 3GC or piperacillin ± tazobactam was not associated with higher mortality, but was associated with increased risk of AmpC overproduction leading to treatment failure compared with cefepime or a carbapenem., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Fecal microbiota transplantation is an effective treatment in recurrent Clostridium difficile infection. Promising results to eradicate multidrug-resistant bacteria have also been reported with this procedure, but there are safety concerns in immunocompromised patients. We report results in ten adult patients colonized with multidrug-resistant bacteria, undergoing fecal microbiota transplantation before (n=4) or after (n=6) allogeneic hematopoietic stem cell transplantation for hematologic malignancies. were obtained from healthy related or unrelated donors. Fecal material was delivered either by enema or via nasogastric tube. Patients were colonized or had infections from either carbapenemase-producing bacteria (n=8) or vancomycin-resistant enterococci (n=2). Median age at fecal microbiota transplantation was 48 (range, 16-64) years. Three patients needed a second transplant from the same donor due to initial failure of the procedure. With a median follow up of 13 (range, 4-40) months, decolonization was achieved in seven of ten patients. In all patients, fecal micro-biota transplantation was safe: one patient presented with constipation during the first five days after FMT and two patients had grade I diarrhea. One case of gut grade III acute graft- versus -host disease occurred after fecal microbiota transplantation. In patients carrying or infected by multidrug-resistant bacteria, fecal microbiota transplantation is an effective and safe decolonization strategy, even in those with hematologic malignancies undergoing hematopoietic stem cell transplantation., (Copyright© 2019 Ferrata Storti Foundation.)

Background: Third-generation cephalosporins (3GCs) are recommended for empirical antibiotic therapy of community-acquired pneumonia (CAP) in patients requiring ICU admission. However, their extensive use could promote the emergence of extended-spectrum beta-lactamases-producing Enterobacteriaceae. Our aim was to assess whether the use of 3GCs in patients with CAP requiring ICU admission was justified., Methods: We assessed all patients with CAP who required ICU admission during a 7-year period. We recorded empirical and definitive antibiotic therapies and susceptibility of causative pathogens. Amoxicillin, amoxicillin/clavulanate (A/C) susceptibilities as well as amikacin susceptibility of A/C-resistant strains were recorded., Results: From January 2007 to March 2014, 391 patients were included in the study. Empirical 3GCs were used in 215 patients (55%). Among 267 patients with microbiologically documented CAP (68%), 241 received a beta-lactam as definitive therapy, and of those, 3CGs were chosen for 43 patients (18%). Amoxicillin or A/C was active against isolated pathogens in 159 patients (66%), while 39 patients (16%) required a beta-lactam with a broader spectrum than 3GCs. Ninety-four per cent of A/C-resistant strains were amikacin susceptible., Conclusions: In ICU patients with CAP, 3GCs given on an empirical basis are changed, according to microbiological documentation, for another beta-lactam in 82% of cases especially to A/C in the absence of resistance risk factor. In patients evidencing risk factors for A/C-resistant strains infection, 3GCs or antipseudomonal beta-lactams including carbapenem associated with amikacin in the most severe patients seem a relevant empirical antibiotic therapy. This strategy could decrease 3GCs' use.

Gardnerella vaginalis is a facultative anaerobic bacterium that inhabits the genitourinary tract of both healthy women and those with bacterial vaginosis. We report a case of G. vaginalis bacteremia associated with severe toxic encephalopathy in a young woman. Anaerobic blood cultures yielded pure growth of small gram-variable rods later identified as G. vaginalis by both rapid biochemical tests and 16S rRNA gene sequencing. The patient recovered after treatment with amoxicillin-clavulanate according to the in vitro susceptibility testing. The complete genome of G. vaginalis isolate from blood cultures was determined. In vitro G. vaginalis isolate produced elevated amounts of a pore-forming toxin vaginolysin compared to control G. vaginalis isolates. We hypothesize that this toxin, if produced in high amounts in blood, is able to disrupt the blood-brain barrier and exert a toxic activity on brain cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: In critically ill patients with pneumonia, accurate microorganism identification allows appropriate antibiotic treatment. In patients undergoing bronchoalveolar lavage (BAL), direct examination of the fluid using Gram staining provides prompt information but pathogen identification accuracy is low. Culture of BAL fluid is actually the reference, but it is not available before 24 to 48 h. In addition, pathogen identification rate observed with direct examination and culture is decreased when antibiotic therapy has been given prior to sampling. We therefore assessed, in critically ill patients with suspected pneumonia, the performance of a multiplex PCR (MPCR) to identify pathogens in BAL fluid. This study is a prospective pilot observation., Methods: We used a MPCR detecting 20 types of microorganisms. Direct examination, culture, and MPCR were performed on BAL fluid of critically ill patients with pneumonia suspicion. The final diagnosis of infective pneumonia was retained after the medical chart was reviewed by two experts. Pathogen identification rate of direct examination, culture, and MPCR in patients with confirmed pneumonia was compared., Results: Among the 65 patients with pneumonia suspicion, the diagnosis of pneumonia was finally retained in 53 cases. Twenty nine (55%) were community-acquired pneumonia and 24 (45%) were hospital acquired. Pathogen identification rate with MPCR (66%) was greater than with culture (40%) and direct examination (23%) (p =0.01 and p <0.001, respectively). When considering only the microorganisms included in the MPCR panel, the pathogen identification rate provided by MPCR reached 82% and was still higher than with culture (35%, p <0.001) and direct examination (21%, p <0.001). Pathogen identification rate provided by MPCR was not modified in the case of previous antibiotic treatment (66% vs. 64%, NS) and was still better than with culture (23%, p <0.001)., Conclusions: The results of this pilot study suggest that in critically ill patients, MPCR performed on BAL fluid could provide higher identification rate of pathogens involved in pneumonia than direct examination and culture, especially in patients having received antimicrobial treatment.

In this prospective multicentric study, we assessed the in vitro antimicrobial activity of carbapenems (imipenem, meropenem, and doripenem), tigecycline, and colistin against 166 unusual nonfermenting Gram-negative bacilli (NF-GNB) clinical isolates collected from nine French hospitals during a 6-month period (from December 1, 2008, to May 31, 2009). All NF-GNB isolates were included, except those phenotypically identified as Pseudomonas aeruginosa or Acinetobacter baumannii. Minimal inhibitory concentrations (MICs) of antimicrobial agents were determined by using the E-test technique. The following microorganisms were identified: Stenotrophomonas maltophilia (n=72), Pseudomonas spp. (n=30), Achromobacter xylosoxidans (n=25), Acinetobacter spp. (n=18), Burkholderia cepacia complex (n=9), Alcaligenes faecalis (n=7), and Delftia spp. (n=5). All isolates of Acinetobacter spp., A. faecalis, and Delftia spp. were susceptible to the three carbapenems. Imipenem exhibited the lowest MICs against Pseudomonas spp., and meropenem, as compared with imipenem and doripenem, displayed an interesting antimicrobial activity against A. xylosoxidans and B. cepacia complex isolates. Conversely, no carbapenem exhibited any activity against S. maltophilia. Except for S. maltophilia isolates, tigecycline and colistin exhibited higher MICs than carbapenems, but covered most of the microorganisms tested in this study. To our knowledge, no prior study has compared antimicrobial activity of these five antibiotics, often considered as "last-resort" treatment options for resistant Gram-negative infections, against unusual NF-GNB clinical isolates. Further studies should be carried out to assess the potential clinical use of these antibiotics for the treatment of infections due to these microorganisms.

Two mutants of Escherichia coli (V1 and V2) with acquired mechanisms of resistance to fluoroquinolones were isolated sequentially from blood cultures of a patient with cholangiocarcinoma treated repeatedly with ofloxacin; a third mutant (V3) was isolated under ciprofloxacin therapy. All mutants were related clonally. V1 was susceptible to quinolones but with diminished susceptibility to ofloxacin. V2 was hypersusceptible to nalidixic acid but had high-level resistance to ofloxacin. V3 was resistant to all quinolones. Ofloxacin selected for original gyrA and parC mutations, leading to the unusual and misleading resistance phenotypes of V1 and V2, whereas efflux played a major role in the increased resistance of V3.

Objective: 1/ To evaluate if empirical antibiotic prescription on admission to our intensive care unit (ICU) respects the local recommendations for antibiotic prescription and to identify predictors of nonadherence to these guidelines. 2/ To assess whether nonadherence to the guidelines is associated with increased in-hospital mortality due to the initial infection., Materials and Methods: This was a prospective six-month observational study performed in a 14-bed medical ICU. Patients were included if they received curative antibiotic therapy on admission. Respect of the local treatment recommendations was evaluated according to adherence to the local empirical guidelines defined in a 80-page booklet which is given in our hospital to every physician., Results: Among 132 antibiotic prescriptions, 21 (16%) were unjustified (absence of infection), 17 (13%) were microbiologically documented at admission, and nine (7%) were given for infections from unknown origin. Among the 85 (64%) empirical prescriptions that could be evaluated for adherence to local recommendations, nine (11%) were inappropriate and 76 (89%) appropriate. In univariate analysis hospital-acquired infection was the sole predictor of inappropriate treatment (p = 0.0475). Independent predictors of in-hospital mortality due to the initial infection were inappropriate empirical treatment (odds ratio [OR] = 14.64, 95% confidence interval [CI]: 2.17-98.97; p = 0.006), prescription of fluoroquinolones (OR = 8.22, 95% CI: 1.88-35.95; p = 0.005) and a higher Simplified Acute Physiology Score II score (per one-point increment (OR = 1.04, 95% CI: 1.01-1.07; p = 0.02)., Conclusion: Nonadherence to local empirical antibiotic therapy guidelines was associated with increased in-hospital mortality due to the initial infection.

Objectives: We previously showed that real-time PCR was a reliable technique for coupled detection of Helicobacter pylori and clarithromycin resistance mutations directly from biopsies. After one year of use, we compared its performances to those of histology, which remains the most employed method for H. pylori detection from gastric biopsies., Materials and Methods: 518 subjects underwent endoscopy during the year 2003 with biopsies taken for H. pylori detection by histology, PCR, and in case of discrepancy between the two techniques, by culture., Results: The prevalence of infection, defined as positive PCR and histology, and in case of discrepancy as a positive culture, was 30% (163/518). The percentage of concordance between the two tests was 87.8% (455/518). The sensitivity, specificity, positive and negative predictive values of PCR were 98.2%, 97.5%, 94.7%, and 99.1%, respectively. The corresponding performances of histology were 87.7%, 91.3%, 82.2%, and 94.2%, respectively (p<0.001). The prevalence of clarithromycin resistance was 30%., Conclusions: PCR is more accurate in routine than histology and permits easy determination of clarithromycin resistance, which is useful in countries like France where the prevalence of resistance is high.

The cdeA gene, cloned from Clostridium difficile clinical strain 714 under the control of its natural promoter made Escherichia coli and Clostridium perfringens resistant to ethidium bromide and acriflavin but had no effect on the susceptibility of the hosts to the following antibiotics: norfloxacin, ciprofloxacin, gentamicin, erythromycin, tetracyclin, and chloramphenicol. However, it was responsible for fluoroquinolone resistance in E. coli when it was cloned under the control of the Plac promoter. Quantitative reverse transcriptase (RT)-PCR showed that growth of C. difficile clinical strain 253 in the presence of subinhibitory concentrations of ethidium bromide significantly increased the transcription of cdeA, but this was not observed with ciprofloxacin. The deduced protein was homologous to the protein sequences of known efflux pumps from the third cluster (the so-called DinF branch) of the multidrug and toxic compound extrusion (MATE) family. CdeA caused ethidium bromide energy-dependent efflux in whole cells of E. coli. Efflux activity was stimulated by addition of Na+ ions, suggesting that CdeA, like other pumps of the MATE family, is a Na+-coupled efflux pump. CdeA is the first multidrug efflux transporter identified in C. difficile., (Copyright Mary Ann Liebert, Inc.)

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.

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.

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 micro g 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 micro g/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.

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- kappa B, 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- kappa B 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.

Helicobacter pylori resistance to macrolides is increasing, and the need for susceptibility testing has become crucial. The only standardized method is agar dilution, which is not adapted to clinical practice. The present work aimed: (1) to optimize the technical conditions and to assess the reproducibility of the E-test and disk diffusion method for macrolides susceptibility testing of H. pylori, and (2) to assess the performances of these two phenotypic methods in detecting strains harboring a resistance mechanism to macrolides. We used 191 isolates collected in nine centers of France and Belgium. Phenotypic tests were performed on Mueller-Hinton agar supplemented with 10% horse blood, inoculated with a 2-day-old H. pylori suspension (10(8) CFU/ml), and incubated for 72 hr at 37 degrees C under microaerophilic conditions. The reproducibility studied on two randomly selected strains was better for disk diffusion than for the E-test for both clarithromycin and erythromycin. For a subset of 10 strains, the MICs of erythromycin and clarithromycin did not differ from more than one two-fold dilution when determined by E-test or agar dilution method. The breakpoints were for MICs: 1 mg/L for both clarithromycin and erythromycin and for inhibition diameters, 22 mm for clarithromycin and 17 mm for erythromycin. There was a 100% concordance between susceptibility to erythromycin and clarithromycin. However, the susceptible and resistant populations were better separated by testing erythromycin. Of 34 resistant strains, two lacked the A2142G and A2143G point mutations in 23S rRNA by PCR-RFLP. None of 15 tested sensitive strains were positive for one of these two point mutations. For clinical practice, we recommend to assess macrolide susceptibility of H. pylori by using one of these two phenotypic methods under the described technical conditions.

Clarithromycin resistance of Helicobacter pylori is relatively frequent in France and is assumed to be the main cause of failure of the proton pump inhibitor-amoxicillin-clarithromycin (PPI-AC) therapy, which is the first-line regimen in our country. We determined the respective effects of clarithromycin primary and secondary resistances on efficacy of the PPI-AC regimen and examined whether failures were associated with persistence of the same strain or with emergence of a new one. Hundred and twenty three H. pylori-infected patients were treated for seven days with omeprazole 20 mg b.d., amoxicillin 1 g b.d., and clarithromycin 500 mg b.d. Eradication was assessed by breath test in 102 patients. MICs of clarithromycin were determined by E-test. Strain genotyping was performed by random amplified polymorphic DNA. The pre-treatment and post-treatment prevalences of clarithromycin resistance were 18.7% (23/123) and 69.2% (9/13), respectively. The rates of eradication were 67.6% (69/102), 78.8% (67/85), and 11.8% (2/17) for all, susceptible and resistant strains, respectively. The post-treatment isolate was available for six patients with a susceptible pre-treatment isolate and a persistent infection; resistance emerged in two patients and was associated with persistence of the pre-treatment strain in one and with selection of a new strain in the other. In conclusion, in our hospital, failures of the PPI-AC therapy are related to both clarithromycin primary and secondary resistances but emergence of secondary resistance does not explain all failures in the initial clarithromycin-susceptible group. In that group a new strain can emerge after failure.

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.