Jean-François Lemeland

Multiplex PCR Targeting tpi (Triose Phosphate Isomerase), tcdA (Toxin A), and tcdB (Toxin B) Genes for Toxigenic Culture of Clostridium difficile

ABSTRACT A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.

Multilocus Sequence Typing Analysis of Human and Animal Clostridium difficile Isolates of Various Toxigenic Types

ABSTRACT A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 72 Clostridium difficile isolates from various hosts, geographic sources, PCR ribotypes, and toxigenic types (determined by PCR targeting tcdA and tcdB genes). MLST was performed by DNA sequence analysis of seven housekeeping genes (aroE, ddl, dutA, tpi, recA, gmk, and sodA). The number of alleles ranged from five (dutA and ddl) to eleven (recA). Allelic profiles allowed the definition of 34 different sequence types (STs). These STs lacked correlation with geographic source but were well correlated to toxigenic type. The dendrogram generated from a matrix of pairwise genetic distances showed that animal isolates did not constitute a distinct lineage from human isolates and that there was no hypervirulent lineage within the population of toxigenic human isolates (isolates recovered from pseudomembranous colitis and antibiotic-associated diarrhea did not cluster in distinct lineages). However, A− B+ variant isolates shared the same ST that appeared as a divergent lineage in the population studied, indicating a single evolutionary origin. The population structure was further examined by analysis of allelic polymorphism. The dendrogram generated from composite sequence-based analysis revealed a homogeneous population associated with three divergent lineages, one of which was restricted to A− B+ variant isolates. C. difficile exhibited a clonal population structure, as revealed by the estimation of linkage disequilibrium (Ia) between loci. The analysis of alleles within clonal complexes estimated that point mutation generated new alleles at a frequency eightfold higher than recombinational exchange, and the congruence of the dendrograms generated from separate housekeeping loci confirmed the mutational evolution of this species.

In vitro synergistic effects of double and triple combinations of beta-lactams, vancomycin, and netilmicin against methicillin-resistant Staphylococcus aureus strains.

ABSTRACT Several studies have previously reported synergistic effects between vancomycin and a given β-lactam or a given aminoglycoside against methicillin-resistant Staphylococcus aureus (MRSA) strains. The aim of our study was to exhaustively compare the effects of different combinations of a β-lactam, vancomycin, and/or an aminoglycoside against 32 clinical MRSA strains with different aminoglycoside susceptibility patterns. The effects of 26 different β-lactam–vancomycin and 8 different aminoglycoside-vancomycin combinations were first studied using a disk diffusion screening method. The best interactions with vancomycin were observed with either imipenem, cefazolin, or netilmicin. By checkerboard studies, imipenem-vancomycin and cefazolin-vancomycin each provided a synergistic bacteriostatic effect against 22 strains; the mean fractional inhibitory concentration (FIC) indexes were 0.35 and 0.46 for imipenem-vancomycin and cefazolin-vancomycin, respectively. The vancomycin-netilmicin combination provided an indifferent effect against all of the 32 strains tested; the mean of FIC index was 1.096. The mean concentrations of imipenem, cefazolin, netilmicin, and vancomycin at which FIC indexes were calculated were clinically achievable. Killing experiments were then performed using imipenem, cefazolin, netilmicin, and vancomycin at one-half of the MIC, alone and in different combinations, against 10 strains. The vancomycin-netilmicin regimen was rarely bactericidal, even against strains susceptible to netilmicin. The imipenem-vancomycin and cefazolin-vancomycin combinations were strongly bactericidal against six and five strains, respectively. The addition of netilmicin markedly enhanced the killing activity of the combination of cefazolin or imipenem plus vancomycin, but only for the MRSA strains against which the β-lactam–vancomycin combinations had no bactericidal effect. It is noteworthy that the latter strains were both susceptible to netilmicin and heterogeneously resistant to methicillin.

Septicemia Due to Pasteurella pneumotropica: 16S rRNA Sequencing for Diagnosis Confirmation

ABSTRACT Bacteremia due to Pasteurella pneumotropica occurs infrequently. We report a case of septicemia in a 72-year-old woman who had no underlying illness. The microorganism was isolated from 10 blood cultures and identified by conventional and molecular methods. This is the first reported case of P. pneumotropica septicemia in an immunocompetent patient. The history of P. pneumotropica diseases in animals and humans and their varied clinical features are reviewed.

Tolerance to the Glycopeptides Vancomycin and Teicoplanin in Coagulase-Negative Staphylococci

ABSTRACT Tolerance to vancomycin and teicoplanin in 90 clinical isolates of coagulase-negative staphylococci (CoNS) was investigated by time-kill curve methodology. Only six strains, belonging to the Staphylococcus lugdunensis species, exhibited tolerance. The seven other S. lugdunensis strains tested displayed weak susceptibility to the bactericidal activity of glycopeptides compared to the other CoNS. These phenomena are of concern, since S. lugdunensis is recognized as one of the most pathogenic CoNS.

Influence of antibiotics and food intake on liver glutathione and cytochrome P-450 in septic rats

Experimental sepsis in rats induces a restriction in spontaneous food intake and a drop in liver glutathione, cytochrome P-450 (P-450) and aminopyrine demethylase (AD) activity. The present study was designed to assess the effects of antibiotics alone or when combined with food deprivation on these variables. Eighty-nine male Sprague-Dawley rats were assigned to six groups: control (C), acute infection (experimental pyelonephritis, I), acute infection with antibiotics and food given ad lib. (IA), control with antibiotics (CA), acute infection with antibiotics pair-fed to I (IAR), and sham-operated pair-fed to I (SR). Liver glutathione, P-450 and AD activities were reduced by 45.2, 79.8 and 41.2% respectively in group I. Glutathione and AD significantly increased only in those infected rats given antibiotics and allowed free access to food. P-450 did not normalize within the study period in infected rats receiving antibiotics and food repletion. The risk of drug hepatotoxicity in acute septic states is therefore closely related to the nutritional status. From this point of view, nutritional support is almost as important as treatment of infection.

Detection of Neisseria meningitidis DNA from skin lesion biopsy using real-time PCR: usefulness in the aetiological diagnosis of purpura fulminans

ObjectiveThe present study evaluated the usefulness of a real-time polymerase chain reaction (rtPCR) assay for the detection of Neisseria meningitidis (Nm) and genogrouping on skin lesion biopsies in patients with purpura fulminans (PF).DesignRetrospective single-centre study.SettingAdult and paediatric intensive care units at the University Hospital of Rouen.PatientsAll patients admitted between January 2000 and January 2006, with a final diagnosis of PF and for which a skin biopsy and blood cultures were performed, were included.InterventionsSkin biopsy and blood cultures were used for culture and rtPCR.Measurements and main resultsThirty-four patients fulfilled the criteria (27 children and 7 adults). Nm rtPCR performed on skin biopsy was positive in 100% (34/34) of cases, compared with only 14.7% (5/34) for skin culture (p = 0.0001). rtPCR genogrouping on skin biopsy was positive in 58.8% (20/34) of the cases compared with 14.7% (5/34) for skin culture (p = 0.0013). For patients (n = 17) in whom rtPCR was performed both on blood and skin biopsy, skin biopsy gave a significantly higher rate of Nm detection [100% (17/17) vs. 58.8% (10/17); p = 0.023] and genogroup characterisation [76.5% (13/17) vs. 35.3% (6/17); p = 0.045] than blood. We encountered no specimen with culture-positive and rtPCR-negative results (negative predictive value of rtPCR 100%).ConclusionIn suspected PF cases, skin biopsy is more reliable to identify Nm and its genogroup than blood or, probably, CSF, especially when PCR methods are used. This could help the implementation of public health interventions, especially concerning a vaccination policy.

Group R streptococci: wild boars as a second reservoir.

Group R streptococci have caused many cases of septicaemia and meningitis in patients handling live or slaughtered pigs which were the only known reservoir of group R streptococci. A human case, due to a wild boar, is reported here and it is therefore concluded that there exist both a domestic (pig) and a wild (wild boar) reservoir of group R streptococci.

Proteomic analysis of Staphylococcus aureus biofilms grown in vitro on mechanical heart valve leaflets.

The in vitro colonization of three commercial heart valve leaflets by Staphylococcus aureus was investigated. The leaflets, made of pyrolytic carbon alloyed with or without silicon, displayed similar surface properties (wettability, roughness) and were readily colonized by S. aureus that formed patchy biofilms on the three supports. A proteomic approach was used to assess the physiological status of biofilm populations by comparing their protein maps to those of bacteria cultured as free cells in the presence or absence of biofilm substratum. Principal component analysis (PCA) revealed, for each tested leaflet, statistical relationships between the protein maps of the biofilm and free-floating microbial populations. A spot-by-spot comparison of protein levels on two-dimensional electropherograms showed that many proteins were accumulated or underproduced by microbial populations grown in the presence of a leaflet compared with protein levels in control free populations. The number of accumulated proteins was noticeably higher than that of underproduced polypeptides. This protein overproduction was emphasized in biofilm populations. Several proteins, some of which were identified, were differentially produced by both surface-associated planktonic and biofilm-grown cell populations compared with control free-cell ones cultured in the absence of leaflet, whatever the leaflet tested. The potential of this proteomic approach for fighting against microbial adhesion and biofilm formation is discussed.

In vitro interactions between different beta-lactam antibiotics and fosfomycin against bloodstream isolates of enterococci.

The effects of 16 different beta-lactam-fosfomycin combinations against 50 bloodstream enterococci were compared by a disk diffusion technique. Cefotaxime exhibited the best interaction. By checkerboard studies, the cefotaxime-fosfomycin combination provided a synergistic bacteriostatic effect against 45 of the 50 isolates (MIC of cefotaxime at which 90% of the isolates were inhibited, >2,048 micrograms/ml; MIC of fosfomycin at which 90% of the isolates were inhibited, 128 micrograms/ml; mean of fractional inhibitory concentration indexes, 0.195). By killing curves, cefotaxime (at 64 micrograms/ml) combined with fosfomycin (at > or = 64 micrograms/ml) was bactericidal against 6 of 10 strains tested.