Highly selective recovery of Ni(II) in neutral and acidic media using a novel Ni(II)-ion imprinted polymer.

In this work, an original ion-imprinted polymer (IIP) was synthetized for the highly selective removal of Ni(II) ions in neutral and acidic media. First a novel functional monomer (AMP-MMA) was synthetized through the amidation of 2-(aminomethyl)pyridine (AMP) with methacryloylchloride. Following Ni(II)/AMP-MMA complex formation study, the Ni(II)-IIP was produced via inverse suspension polymerization (DMSO in mineral oil) and characterized with solid state 13C CPMAS NMR, FT-IR, SEM and nitrogen adsorption/desorption experiments. The Ni(II)-IIP was then used in solid-phase extraction of Ni(II) exploring a wide range of pH (from neutral to strongly acidic solution), several initial concentrations of Ni(II) (from 0.02 to 1 g/L), and the presence of competitive ions (Co(II), Cu(II), Cd(II), Mn(II), and Mg(II)). The maximum Ni(II) adsorption capacity at pH 2 and pH 7 reached values of 138.9 mg/g and 169.5 mg/g, that are among the best reported in literature. The selectivity coefficients toward Cd(II), Mn(II), Co(II), Mg(II) and Cu(II) are also very high, with values up to 38.6, 32.9, 25.2, 23.1 and 15.0, respectively. The Ni(II)-IIP showed good reusability of up to 5 cycles both with acidic and basic Ni(II) eluents.

PlaScope: a targeted approach to assess the plasmidome from genome assemblies at the species level.

Plasmid prediction may be of great interest when studying bacteria of medical importance such as Enterobacteriaceae as well as Staphylococcus aureus or Enterococcus. Indeed, many resistance and virulence genes are located on such replicons with major impact in terms of pathogenicity and spreading capacities. Beyond strain outbreak, plasmid outbreaks have been reported in particular for some extended-spectrum beta-lactamase- or carbapenemase-producing Enterobacteriaceae. Several tools are now available to explore the 'plasmidome' from whole-genome sequences with various approaches, but none of them are able to combine high sensitivity and specificity. With this in mind, we developed PlaScope, a targeted approach to recover plasmidic sequences in genome assemblies at the species or genus level. Based on Centrifuge, a metagenomic classifier, and a custom database containing complete sequences of chromosomes and plasmids from various curated databases, PlaScope classifies contigs from an assembly according to their predicted location. Compared to other plasmid classifiers, PlasFlow and cBar, it achieves better recall (0.87), specificity (0.99), precision (0.96) and accuracy (0.98) on a dataset of 70 genomes of Escherichia coli containing plasmids. In a second part, we identified 20 of the 21 chromosomal integrations of the extended-spectrum beta-lactamase coding gene in a clinical dataset of E. coli strains. In addition, we predicted virulence gene and operon locations in agreement with the literature. We also built a database for Klebsiella and correctly assigned the location for the majority of resistance genes from a collection of 12 Klebsiella pneumoniae strains. Similar approaches could also be developed for other well-characterized bacteria.

Intrapartum group B Streptococcus screening in the labor ward by Xpert® GBS real-time PCR.

Group B Streptococcus (GBS) is the leading cause of neonatal infections in industrialized countries. Intrapartum antibiotic prophylaxis (IAP) given to colonized parturients is a key step for the prevention of neonatal early-onset infection. We compared the performances of Xpert® GBS polymerase chain reaction (PCR) (Cepheid, Sunnyvale, CA, USA) as a point-of-care system in labor wards to standard culture for intrapartum GBS detection. Pregnant women with a GBS-positive antenatal screening were prospectively included. A vaginal double swab was collected at the time of delivery for point-of-care Xpert® GBS PCR and GBS culture. A total of 565 pregnant women were included. Valid Xpert® GBS results were obtained for 488 (86.4%) women on the first attempt. Repeat testing improved the PCR success to 516 (91.3%) women. Among the 305 women positive for GBS by culture at delivery, only 238 (78.0%) were positive by Xpert® GBS PCR, cycle thresholds being correlated to culture quantification. Among 260 women negative for GBS culture, 56 (21.5%) were positive by Xpert® GBS PCR, including 50 where IAP was initiated before vaginal sampling. Overall, among the 565 women with GBS antenatal positive culture, only 335 (59.3%) were still positive at delivery whatever the technique used, resulting in unnecessary IAP for 40% of them. This large cohort study comparing intrapartum to antepartum GBS detection provides evidence that (i) Xpert® GBS PCR might be a valuable solution for intrapartum GBS detection compared to culture-based strategies and (ii) laboratory training of non-specialized staff is mandatory to reach the performances required for point-of-care tests.

Bacteraemia caused by third-generation cephalosporin-resistant Escherichia coli in France: prevalence, molecular epidemiology and clinical features.

Escherichia coli is one of the major pathogens responsible for bactaeremia. Empirical antibiotherapy of these infections usually relies on third-generation cephalosporins (3GCs). Thus, the occurrence and epidemiology of 3GC-resistant strains have to be monitored. The French prospective multicentre study COLIBAFI collected 1081 strains of E. coli responsible for bacteraemia in 2005. In the present work, the prevalence of resistance to 3GCs was evaluated, and the implicated molecular mechanisms were characterized by specific PCR and sequencing. Phylogenetic grouping, O-typing, pulsed-field gel electrophoresis and virulence factor analysis were used to investigate the genetic background of the 3GC-resistant (3GC-R) strains. Clinical features of the patients with documented data (n = 1051) were analysed. Decreased susceptibility to 3GCs was observed in 41 strains (3.8%): 19, 18 and four had extended-spectrum ß-lactamase (ESBL), AmpC cephalosporinase and OXA-type penicillinase phenotypes, respectively. Pulsed-field gel electrophoresis revealed that the 3GC-R strains constitute a diverse population. All but one of the strains with an ESBL phenotype produced a CTX-M-type enzyme, and six of them belonged to the widespread intercontinental clone O25b:H4-ST131. AmpC phenotype strains harboured various chromosomal ampC promoter and coding region mutations and/or the bla(CMY-2) plasmidic gene. 3GC-R strains carried fewer virulence factors and were more co-resistant to other antibiotics than 3GC-susceptible (3GC-S) strains. Infections with 3GC-R strains were mostly community-acquired and, as compared with those caused by their 3GC-S counterparts, were more severe. Underlying chronic disease and prior use of antibiotics were independent risk factors for development of a 3GC-R strain bacteraemia. The fact that the molecular support of 3GC resistance is mainly plasmid-mediated represents a potentially epidemic threat.

Synthetic peptide issued from Hap1/LipL32 for new early serodiagnosis of human leptospirosis.

Leptospirosis is a worldwide zoonosis. Today, serological diagnosis is generally assessed by MAT. We performed ELISA with a synthetic peptide derived from Hap1/lipL32 which is a protein expressed only by pathogenic Leptospira. Repeatability and thresholds were defined with 85 controls sera and 119 hospitalized leptospirosis. The PP-ELISA repeatability and IgM/IgG cut-off values were based on control sera. For these cut-off values, we observed the IgM-PP-ELISA specificity of 89%, whereas it was 100% for the IgG. Then, we compared PP-ELISA and standard MAT results for leptospirosis patients. The concordance rate for IgM-PP-ELISA and MAT was low (43%), whereas it was 85% for IgG-PP-ELISA and MAT. During the first 5 days after hospitalization, PP-ELISA gave positive results in 13 out of 16 patients (81%) whereas 8 out of 14 patients (57%) were positive to MAT. ELISA using Hap1/lipL 32-derived synthetic peptide PP is an earlier serological diagnosis of human leptospirosis than MAT.

Leptospira exposure in the human environment in France: A survey in feral rodents and in fresh water.

This paper confirms the important role of rodents to be maintenance hosts of leptospires. Their role is related to renal carriage and shedding of leptospires into urine, thus contaminating fresh water. Serological and carriage of feral rodents trapped in France were determined by MAT and hap1PCR specific for pathogenic leptospires. In same areas, fresh water samples were analyzed by hap1PCR. The overall seroprevalence was 44% in 649 rodents and was similar regardless of the species. Seroprevalence for leptospirosis is about 20-53% according to species. hap1PCR (516 kidneys) showed that renal carriage was higher in brown rats (34.7%) and muskrats (15.8%) than in coypus (3.3%). hap1PCR demonstrates a significative difference (P-value > 10(-12)) for the renal carriage between the different species: muskrats and rats are more efficient maintenance hosts than coypu but all infect water. Moreover 5/38 water samples associated with human cases were hap1PCR positive and 1/113 in controlled waters.

[Evaluation of the impact of different lengths of pre-enrichment in a nutritive broth and prolonged incubation of MRSA-ID, a chromogenic agar medium, on its performances for identifying methicillin-resistant Staphylococcus aureus in screening samples]. / Evaluation de l'influence des conditions de culture sur les performances du milieu gélosé sélectif MRSA-ID pour l'identification de Staphylococcus aureus résistant à la méticilline dans les prélèvements de dépistage.

MRSA-carrier screening is recommended to prevent MRSA dissemination in hospitals. Rapid and specific detection of MRSA in the laboratory is a key element in enabling control measures. Our objective was to evaluate the impact of different lengths of pre-incubation in a nutritive broth and prolonged incubation of MRSA-ID, a chromogenic agar medium, on its performances for identifying MRSA in screening samples. According to our results, short-length pre-enrichments only provided a weak increase of sensitivity as compared to the absence of pre-enrichment. On the contrary, the sensitivity increase provided by an overnight pre-enrichment was significant. The prolongation of incubation in the chromogenic agar medium (48 hours instead of 24 hours) did not provide any significant increase of sensitivity but was associated with a strong and significant loss of specificity. Therefore, it seems relevant to reject prolonged incubation of selective agar media and to make a choice between the absence of pre-enrichment (faster results) and an overnight pre-enrichment (higher sensitivity), according to local epidemiology and local practices implemented for prevention.

[Using a 48-hour delay from admission to the first positive culture without any other consideration: an accurate method to differentiate acquired and imported methicillin-resistant Staphylococcus aureus?]. / Retenir un délai de 48 heures entre l'admission des patients et le premier prélèvement positif est-il suffisant pour différencier les souches acquises à l'hôpital et importées de Staphylococcus aureus résistant à la méticilline?

To differentiate imported and acquired strains of methicillin-resistant Staphylococcus aureus (MRSA), a 48-hour delay from hospital admission to the first MRSA-positive culture is usually considered. To assess if taking into account this delay without any other consideration is an accurate method, we defined 3 situations for whom we considered the MRSA acquisition status as questionable. The other situations were defined as either acquired MRSA or imported MRSA. We determined the acquisition status of MRSA (acquired, imported, or questionable) isolated during a 20-month period by considering or not considering screening samples performed on admission. The ratio "imported MRSA/acquired MRSA" (I/A) was calculated according to (1) the consideration of MRSA with questionable status as imported or acquired, and (2) the consideration of screening samples or not in the calculation of the ratio. The acquisition status in our hospital was questionable in 3.6% of patients when all samples were considered and in 12,0% when only clinical samples were taken into account (p = 0,01). The ratio I/A was 4-fold higher by considering both clinical and screening cultures and questionable status as imported than by considering only clinical samples and questionable status as acquired. Using a 48-hour delay without any other consideration is probably an accurate method to differentiate acquired and imported MRSA when a selective screening programme at admission in operational. Conversely, this definition seems to be more hazardous in the absence of screening.

Impact of selective screening in the emergency department on meticillin-resistant Staphylococcus aureus control programmes.

Identification of the meticillin-resistant Staphylococcus aureus (MRSA) reservoir by active screening followed by the implementation of contact precautions is one of the major components of MRSA control programmes. The objective of this study was to evaluate the results of a programme of selective screening in an emergency department (ED) and the appropriateness of the contact precautions implemented. This was estimated by distinguishing necessary and unnecessary days of contact precautions. This estimation was performed for all days of contact precautions and, more specifically, for days of preventive contact precautions implemented before the availability of screening results. During a three-year period, screening of MRSA carriers was performed on 0.95% (N=605) of patients visiting the emergency ward. Among the 193 (31.9%) MRSA carriers identified, 159 were hospitalized in the short-length-hospitalization area (SLHA) of the ED and/or in other wards. Among the 140 patients admitted to the SLHA, 44 were hospitalized for at least 48 h, with a mean length of hospitalization of 5.9 days. The cumulative duration of hospitalization of carriers identified by screening was 1897 days. In total, 2370 days of contact precautions (including 924 days of preventive precautions) were implemented for patients screened in the ED. Considering the whole hospital, the appropriateness of this entire programme of contact precautions for patients screened in the ED was 80.0% (52.1% for the SLHA), whereas the specific appropriateness of preventive isolation days was 48.6% (43.6% for the SLHA). This study underscores the risk of MRSA cross-transmission in the SLHA, and the usefulness of implementing a control programme of screening carriers in the ED.

Brain abscess caused by Nocardia cyriacigeorgica in a patient with human immunodeficiency virus infection.

Nocardia cyriacigeorgica is a recently characterized species within the genus of Nocardia. We report a brain abscess, following a primary pulmonary colonization, due to this species in a human immunodeficiency virus-infected patient. This case confirms that isolation of Nocardia in sputum is associated with a high risk of disseminated infection in immunocompromised patients.

[Consequences of replacing abacavir for indinavir in successful antiretroviral treatment of a patient with HIV infection]. / Conséquences de la substitution de l'indinavir par l'abacavir chez un patient infecté par le VIH en succès thérapeutique.

INTRODUCTION: Simplification of combined antiretroviral therapy in HIV-infected patients is possible, but virological success can be compromised by the development or emergence of resistant viruses. CASE: Worsening renal functioning in a patient under successful combination antiretroviral therapy resulted led to the replacement of indinavir by abacavir. Eight weeks later, his viral load rose and he developed a mutant virus resistant to all the nucleoside analogs. DISCUSSION: Our case report illustrates the danger of streamlining combined antiretroviral therapy composed only of nucleoside analogs in patients already successfully treated with nucleoside analogs, by exposing them to the risk of the emergence of a mutant virus.

Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1.

The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.

Fluorimetric determination of aluminium in water by sequential injection through column extraction.

A fluorimetric procedure for the determination of aluminium with matrix removal in drinking water is proposed. The system is based both on the solid phase extraction of aluminium on a new chelating resin (XAD-4 modified by grafting salicylic acid) and the fluorimetric detection of a complex formed between 8-hydroxyquinoline-5-sulfonic acid (HQS) and Al(III), after elution of the resin by hydrochloric acid. The sorption and elution of aluminium were studied in both competitive and noncompetitive conditions, varying pH, flow-rates, volume and concentration of reagents, as well as time contact. The optimised procedure allows determination of Al3+ at the sub-ppb level (LOD: 0.2 microg L(-1) for 1 ml of sample) within a working range of 0.2-500 microg L(-1). The analytical procedure was successfully employed for the determination of aluminium in drinking water during and after flocculation/coagulation treatment processes.

Comparison of the efficacy of three commercial bacterins in preventing canine leptospirosis.

Twenty-four specific pathogen-free beagles were randomly allocated into four groups (three vaccinated groups and one control group) and inoculated at nine and 12 weeks of age with one of three commercial inactivated Leptospira vaccines: A (Vanguard 7; Pfizer Santé Animale), B (Dohyvac 7L; Fort Dodge), and C (Nobivac DHPPi + Lepto; Intervet International); the control group received Nobivac DHPPi (Intervet International). Seven weeks after the second vaccination all the dogs were challenged with Leptospira interrogans serogroup canicola. All the vaccinated dogs developed a mild serological response (microscopic agglutination titres) after the booster vaccination. A significant serological response after the challenge was observed, particularly in the controls. The challenge induced fever and clinical disorders in the control group, whereas in the vaccinated groups the clinical signs were mild. Blood cultures became positive in all control dogs, and in one of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B; none of the six dogs vaccinated with vaccine C was leptospiraemic at any stage of the experiment. Urine cultures were positive in all the control dogs two weeks after the challenge. One of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B shed bacteria in their urine after the challenge, but none of the dogs vaccinated with vaccine C shed bacteria in their urine at any time during the experiment.

Nosocomial and community-acquired spontaneous bacterial peritonitis: comparative microbiology and therapeutic implications.

In order to compare the microbiological characteristics of nosocomial and community-acquired episodes of bacterial peritonitis, 95 consecutive, spontaneous episodes were reviewed. Seventy of these episodes were bacteriologically documented. Fifty-three (55.8%) episodes were nosocomial and 42 (44.2%) were community acquired. A total of 78 pathogens were isolated, including 40 gram-positive cocci (34 streptococci, 6 Staphylococcus aureus), 35 gram-negative bacilli (including 23 Escherichia coli), 2 gram-positive bacilli and 1 yeast. Streptococci were found more frequently in community-acquired episodes (53.8%) than in nosocomial episodes (33.3%). Gram-negative bacilli were significantly more frequent in nosocomial episodes than in community-acquired episodes (56.4% vs. 33.3%, P<0.05). Nosocomial isolates were significantly more resistant to amoxicillin-clavulanic acid (48.7% vs. 18.4%, P<0.01) and cefotaxime (33.3% vs. 13.2%, P<0.05) than community-acquired isolates, but no difference was detected regarding resistance to ciprofloxacin. The results indicate that the empirical treatment of spontaneous bacterial peritonitis should differ for nosocomial and community-acquired cases.

Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination.

New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31- to 34-kDa protein fraction of Leptospira interrogans serovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membrane Leptospira protein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologous Leptospira challenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.

Evidence of cross-protection within Leptospira interrogans in an experimental model.

Killed whole-cell preparations were used as bacterins against leptospirosis. As this type of protection is considered to be serogroup-specific, several serogroups were added to the usual vaccines, and the most pathogenic serovar was chosen for each group. Different leptospire extracts were evaluated for their protective capacity against acute lethal leptospirosis in gerbils (Meriones unguiculatus). Total extracts induced complete protection against homologous challenges and partial protection against heterologous challenges. LPS fractions protected against homologous but not heterologous challenges, whereas protein extract induced significant protection against both types of challenge. Thus, cross-protection within L. interrogans was related to the protein extract.

TEM-24 extended-spectrum beta-lactamase-producing Enterobacter aerogenes: long-term clonal dissemination in French hospitals.

OBJECTIVE: To investigate interstrain relatedness of TEM-24-producing Enterobacter aerogenes clinical strains isolated between 1993 and 1998 in 10 French hospitals from nine areas by pulsed-field gel electrophoresis (PFGE) and plasmid patterns. METHODS: Fifteen TEM-24-producing strains and a set of 16 control strains having various other antibiotic resistance phenotypes were genotyped by PFGE. Plasmid DNA from TEM-24-producing strains and transconjugants was analyzed. RESULTS: Analysis of XbaI macrorestriction patterns revealed only minor variations, and showed that all 15 TEM-24-producing strains were closely related. Some isolates originating from distant areas had indistinguishable patterns. According to their clustering correlation coefficients, they were also genomically distant from the control strains. Two plasmid patterns were observed in TEM-24-producing strains, one of them in 13 of the strains. Large plasmids of 85 kb encoding TEM-24 beta-lactamase were present in all isolates and, in all except one strain, could be transferred with high frequency by conjugation. CONCLUSIONS: These results confirm that the spread of the TEM-24 extended-spectrum beta-lactamase in France was essentially due to the dissemination of a single clone.