Rapid diagnosis of Clostridium difficile infection by multiplex real-time PCR.

The gold standards for the diagnosis of Clostridium difficile infections (CDIs) are the cytotoxicity assay and the toxigenic culture. However, both methods are time-consuming and the results are not available before 24-48 h. We developed and evaluated a multiplex in-house real-time polymerase chain reaction (PCR) assay for the simultaneous detection of toxigenic strains of C. difficile and the presumptive identification of the epidemic NAP1/027/BI strain from stools. Amplifications were performed using specific primers for tcdB and tcdC on an ABI Prism 7300 (Applied Biosystems). The detection of amplicons was done using TaqMan probes. The analytical sensitivity of the multiplex real-time PCR for detecting tcdB was estimated to 10 CFU/g of stools. This assay was assessed from 881 consecutive unformed stools from patients suspected of having CDI. The gold standard was the toxigenic culture for the diagnosis of CDI and PCR ribotyping for the identification of the NAP1/027/BI strain. The prevalence of positive toxigenic culture was 9.31%. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 86.59%, 97.43%, 78.02%, and 98.57%, respectively, for the real-time PCR and 70.73%, 100%, 100%, and 97.08%, respectively, for the cytotoxicity assay.

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 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.

ß LACTA test performance for detection of extended-spectrum ß-lactamase-producing Gram-negative bacilli directly on bronchial aspirates samples: a validation study.

OBJECTIVES: Incidence of extended-spectrum ß-lactamase-producing Gram-negative bacilli (ESBL-PE-GNB)-related infections is worryingly increasing worldwide. ESBL-PE-GNB detection directly on bronchial aspirate samples (BAS) performed for suspected pneumonia may help save empirical carbapenems. Our objectives were to optimize ß-LACTA™ test (BLT) realization and evaluate BLT performance for ESBL-PE-GNB detection directly on BAS. METHODS: We studied BLT technical optimization using BAS of different matrix types spiked with increasing concentrations of CTX-M-15-producing Klebsiella pneumoniae; in vitro validation of BLT diagnostic performance on 17 ESBL enzymes, belonging to CTX-M, SHV, TEM, OXA, GES, VEB and PER groups; and clinical validation of BLT performance on 126 BAS prospectively collected from seven intensive care units. RESULTS: After optimization, BLT detected with 100% sensitivity the presence of CTX-M-15-producing K. pneumoniae spiked in sterile BAS for inoculums upon two or more GNB per field upon microscopic Gram staining examination (MGSE). The BLT accurately detected the 17 ESBLs tested at 106 CFU/mL and all ESBLs except Pseudomonas aeruginosa-related OXA-14 at 104 CFU/mL. Among the 126 BAS of the validation cohort, 21 (17%) gave positive BLT (ten in BAS positive and 11 in BAS negative on MGSE). All BLT-positive BAS grew with ESBL-PE-GNB, including five hyper-L2-producing Stenotrophomonas maltophilia strains. BLT detected ESBL-PE-GNB directly on clinical BAS positive for GNB on MGSE and/or growing with ≥104 CFU/mL with 100% sensitivity, specificity, and positive and negative predictive values. CONCLUSIONS: BLT is an accurate tool for ESBL-PE-GNB detection directly on BAS. Further studies are needed to evaluate the impact of BLT-guided early antimicrobial de-escalation strategies.

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 (4 ml kg(-1)) led to the expression of the calcium-independent inducible nitric oxide synthase (iNOS) after 4 h in the duodenum, determined as [14C]-arginine conversion to citrulline. 4. This iNOS activity was not reduced by pretreatment with anti-neutrophil serum (0.4 ml kg(-1), i.p., 3 h before challenge). However, dexamethasone pretreatment (1 mg kg(-1), i.v., 2 h before the extract), or administration of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 5 mg kg(-1), i.v., 2.5 h after the extract) reduced this activity. 5. Furthermore, iNOS was expressed in duodenal isolated epithelial cells 4 h 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 (1 mg kg(-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.

Impact of Helicobacter pylori resistance to clarithromycin on the efficacy of the omeprazole-amoxicillin-clarithromycin therapy.

BACKGROUND: Helicobacter pylori resistance to clarithromycin is relatively frequent in France and is assumed to be the main cause of failure of the proton pump inhibitor-amoxicillin-clarithromycin (proton pump inhibitor-AC) therapy, which is the first-line regimen in France. AIM: To determine the respective effects of clarithromycin primary and secondary resistances on efficacy of the proton pump inhibitor-AC regimen and to determine whether failures are associated with persistence of the same strain or with emergence of a new one. METHODS: A total of 123 H. pylori-infected patients were treated for 7 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. Minimal inhibitory concentrations of clarithromycin were determined by E-test. Strain genotyping was performed by random amplified polymorphic DNA. RESULTS: The pre-treatment and post-treatment prevalences of clarithromycin resistance were 19% (23 out of 123) and 69% (nine out of 13), respectively. The rates of eradication were 68% (69 out of 102), 79% (67 out of 85), and 12% (two out of 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. CONCLUSIONS: In our hospital, failures of the proton pump inhibitor-AC therapy are related to both clarithromycin primary and secondary resistances, but the emergence of secondary resistance does not explain all of the failures in the initial clarithromycin-susceptible group. In that group a new strain can emerge after failure.

CdeA of Clostridium difficile, a new multidrug efflux transporter of the MATE family.

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.

Analysis of the mutations involved in fluoroquinolone resistance of in vivo and in vitro mutants of Escherichia coli.

We searched for the mutations involved in high-level fluoroquinolone resistance (ciprofloxacin MIC > or = 8 microg/ml) of 11 clinical isolates of Escherichia coli. trans-Complementation tests with the wild-type gyrA and parC genes were positive for all strains whereas negative results were observed with the wild-type gyrB and parE genes. By PCR and sequencing, two mutations in gyrA, leading to Ser-83 --> Leu and Asp-87 --> Asn (7) or Gly (2) or Tyr (1) changes, were found in 10 strains, the eleventh presenting only the Ser-83 --> Leu change. In addition, all strains carried one change in ParC: Ser-80 --> Ile (8) or Arg (2); Glu-84 --> Lys (1). We described a novel and simple method permitting detection of the mutations in parC at codon 80, PCR-RFLP with HaeII. In vitro mutants, selected with ciprofloxacin in three successive steps were also studied. The first-step mutants were complemented by pJSW101 (gyrA+) but not by pEN260 (parC+), whereas the second-step and third-step mutants were complemented by both plasmids. Mutations occurred in the following order: (i) gyrA at codon 83 (Ser to Leu change), (ii) parC at codon 80 (Ser to Ile change), and (iii) gyrA at codon 87 (Asp to Asn change). Thus, these sequential mutations appear to be frequently involved in high-level fluoroquinolone resistance of E. coli.

Quinolone resistance mutations in the gyrA gene of clinical isolates of Salmonella.

S. typhimurium AlhR, S. enteritidis OulR, and S. hadar GueR resistant to fluoroquinolones (QR), ciprofloxacin MICs, 0.25 to 1 microgram/ml; norfloxacin MICs, 0.5 to 4 micrograms/ml; nalidixic acid MIC, 256 micrograms/ml were isolated from urinary tract infections (AlhR and OulR) during FQ therapy in immunocompromised patients infected by the parent FQ-susceptible strains (AlhS and OulS) (ciprofloxacin MICs, 0.032-0.063; norfloxacin MICs, 0.125-0.25; nalidixic acid MICs, 4-8) or from intestinal infection (GueR). Transformation of AlhR, OulR, and GueR by plasmid pJSW101 carrying the wild-type gyrA gene of Escherichia coli resulted in complementation (nalidixic acid MICs, 4 to 8), proving that these strains had a gyrA mutation. A 800-bp fragment of gyrA from the five strains was amplified by PCR. Direct DNA sequencing of 252-bp region of this fragment identified a single point mutation leading to a substitution Ser-83 to Tyr in AlhR and to a substitution Ser-83 to Phe in OulR and in GueR. These results emphasize the potential risk of selection of FQ-resistant Salmonella during FQ therapy in immunocompromised patients and suggest that these strains differ from the parent strains at least by one mutation in the gyrA gene. They also confirm the role of substitutions in position 83 of gyrA in FQ-resistant clinical isolates of Salmonella.

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.

Validation of diffusion methods for macrolide susceptibility testing of Helicobacter pylori.

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.

Co-detection of Helicobacter pylori and of its resistance to clarithromycin by PCR.

Our aim was to develop a rapid molecular test based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and making it possible to detect Helicobacter pylori directly from gastric biopsy samples, and to test its susceptibility to clarithromycin. A 629-bp fragment of the 23S rRNA gene of H. pylori was amplified by PCR and the mutations responsible for clarithromycin resistance were detected with Bsa1 and Bbs1 restriction endonucleases. Thirty-five gastric samples were tested in parallel by standard microbiologic methods (culture and clarithromycin susceptibility testing with E-test strips) and by PCR-RFLP. The 10 culture-negative samples were also PCR-negative. Sixteen out of the 25 culture-positive samples (64%) were PCR-positive. RFLP analysis could be done in 12 cases and the results were in agreement with those of the E-test: susceptibility in five cases, resistance in seven (six A2144G mutations and one A2143G mutation).

Construction of a gyrA plasmid for genetic characterization of fluoroquinolone-resistant Staphylococcus aureus.

We have constructed a gyrA trans-complementation plasmid, pAT512, by cloning the wild-type gyrA gene of Staphylococcus aureus into the expression vector pAT392. Introduction by electrotransformation of pAT512 into a high-level fluoroquinolone resistant mutant of S. aureus (ciprofloxacin MIC = 16 micrograms ml-1) having a gyrA mutation which results in a Ser-84 to Leu substitution, reduced the MICs of norfloxacin and ciprofloxacin for the host of four- and eight-fold, respectively.

[Comparison of quantifying Helicobacter pylori gastric infection by culture, histology and C13 urea breath test]. / Comparaison de la quantification de l'infection gastrique à Helicobacter pylori par culture, histologie, et test respiratoire à l'urée marquée au 13C.

OBJECTIVES: In Helicobacter pylori infection, the bacterial burden may play a role in the pathogenesis of gastric or duodenal ulcerated lesions. It could also influence the results of antimicrobial therapy. No simple test has been validated to quantify Helicobacter pylori density. The aim of this study was to determine the value of histology and/or 13C-urea breath test to quantify the infection as compared with quantitative culture, taken as a reference method. PATIENTS AND METHODS: Biopsies samples were taken from the antrum at endoscopy in 72 patients. Thirty-seven patients with positive urease test at 20 minutes were enrolled in the study. Bacterial density was evaluated from biopsies by quantitative culture and semi-quantitative histological examination (score from 0 to 3). The bacterial density was evaluated as well by 13C-urea breath test from the proportion of 13CO2 in exhaled air (delta 13CO2) at 20, 40, and 60 minutes as compared with the basal level. RESULTS: The bacterial density, evaluated by quantitative culture ranged from 5 CFU to 110,000 CFU per mg of tissue. By histology, a score 1 was found in 5 patients, a score 2 in 17, and a score 3 in 15. delta of 13CO2 measured by 13C-urea breath test ranged from 0.2 to 117.5, from 0.2 to 102, and from 0.6 to 66.7 at 20, 40 and 60 minutes respectively. The quantity of bacteria measured by culture was not significantly higher for these with a score of 3 as compared with those with a pooled score of 1 and 2 (P < 0.05). No significant correlation was found between the results of quantitative culture and these of breath test. CONCLUSION: In practice, evaluation of bacterial burden by a histological score seems only accurate for the most severe density (score 3). The 13C-urea breath test does not allow a reliable quantitative evaluation.

[Antibiotic-impregnated prostheses: eclectic indications]. / Prothèses imprégnées d'antibiotiques: des indications éclectiques.

OBJECTIVE: Infection is a major complication in vascular stents. Stents impregnated with gelatine and dipped in Rifampicin have been shown to resist methicillin-resistant Staphylococcus aureus in both animal experiments and in man. It has been suggested that all aorto-ilio-femoral stents should be treated. To evaluate this method, we reassessed all stent infections observed in our patients who had undergone revascularization of the lower limbs from January 1985 to 1994. We excluded stents implanted for ruptured aneurysms or implanted in patients with a past history of local infection on vascular stents. RESULTS: The rate of septic complications observed during the first year was 1% for all patients in the series, 0% for aorto-aortic and aorto-biiliac stents and 0.7% for aorto- bifemoral stents. These rates are similar to those reported in the multicentric study directed by Goeau Brissonière using antibiotic impregnated stents. The extra cost involved in using such stents for aorto-ilio-femoral revascularization was estimated in this series at 2,180,000 Francs. The costs resulting from the three infections was estimated at 960,000 Francs. CONCLUSION: Based on the findings in this series, antibiotic impregnated stents should be indicated only in selected patients due to the extra cost: past history of local infection, ruptured aneurysms, femoro-tibial stents, cross or axillo-femoral revascularization for which the rate of stent infection is 6.3 - 3.2 and 1.4%, immunodeficient patients, multiple reoperations, post-irradiation arteritis and situations known to involve major risk of infection.

Gram-negative bacteremia: which empirical antibiotic therapy?

PURPOSE: Given the increasing frequency of cefotaxime-resistant strains, third-generation cephalosporins (3GC e.g. cefotaxime, ceftriaxone) might not be recommended any longer as empirical antibiotic therapy for community-acquired Gram-negative bacteremia (CA-GNB). PATIENTS AND METHODS: We conducted a multicenter prospective descriptive study including patients with CA-GNB. RESULTS: Two hundred and nineteen patients were included. Escherichia coli and Pseudomonas aeruginosa were the most frequently isolated species in 63% (n=138) and 11% (n=24) of the cases, respectively. The prevalence of cefotaxime-resistance reached 18% (n=39) mostly due to intrinsic resistance (27 cases, 12%). The presence of invasive material (P<0.001), the origin of the patient (Paris region or West of France) (P=0.006), and home health care (P<0.001) were variables predicting resistant GNB. The negative predictive value for resistance in patients with invasive material coming from the West of France, or without invasive material and with home health care was 94%. The positive predictive value for patients with invasive material living in Paris, or without invasive material and with home health care only reached 58 and 54%, respectively. CONCLUSIONS: Using 3GC for CA-GNB due to cefotaxime-resistant strains was relatively frequent, ESBL-producing Enterobacteriaceae being rarely involved. Our study highlights the role of local epidemiology; before any changes to first-line antibiotic therapy, local epidemiological data should be taken into account.