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The significance of wastewater treatment lagoons (WWTLs) as point sources of clinically relevant Pseudomonas aeruginosa that can disseminate through rural and peri-urban catchments was investigated. A panel of P. aeruginosa strains collected over three years from WWTLs and community-acquired infections was compared by pulsed field gel electrophoresis (PFGE) DNA fingerprinting and multilocus sequence typing (MLST). Forty-four distantly related PFGE profiles and four clonal complexes were found among the WWTL strains analyzed. Some genotypes were repeatedly detected from different parts of WWTLs, including the influent, suggesting an ability to migrate and persist over time. MLST showed all investigated lineages to match sequence types described in other countries and strains from major clinical clones such as PA14 of ST253 and "C" of ST17 were observed. Some of these genotypes matched isolates from community-acquired infections recorded in the WWTL geographic area. Most WWTL strains harbored the main P. aeruginosa virulence genes; 13% harbored exoU-encoded cytoxins, but on at least six different genomic islands, with some of these showing signs of genomic instability. P. aeruginosa appeared to be highly successful opportunistic colonizers of WWTLs. Lagooning of wastewaters was found to favor dissemination of clinically relevant P. aeruginosa among peri-urban watersheds., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most outbreaks are linked to contaminated hot water systems and cooling towers. Our study was about the molecular typing of 35 strains of L. pneumophila including four clinical isolates and 31 environmental strains isolated from the distribution systems of 14 hotels. Among the clinical strains, two have the same pattern, however, all were different from the studied environmental strains. For the 31 environmental strains, ten patterns were obtained. Among which, a same pulsotype was found for four strains isolated from four different establishments. In addition, two different pulsotypes were found for strains isolated from the same establishment. The pulsed-field gel electrophoresis showed the existence of various patterns. Although cases of legionellosis were declared in these hotels, there are no epidemiological links between the clinical and environmental strains.

There are currently more than 40 species of Legionella and the identification of most of them by standard methods is often technically difficult. The aim of this study was to use a ribotyping method with endonuclease HindIII and a probe consisting of a set of five oligonucleotides (referred to as OligoMix5). A total of 123 strains, including 78 type or reference strains corresponding to 44 species, eight clinical and 37 environmental isolates were tested. The usefulness of the method was demonstrated for the identification at the species level of all of the 123 Legionella isolates tested, with each species showing a specific profile. Among the 15 serogroups of Legionella pneumophila, eight patterns were obtained. For the 45 field strains, the randomly amplified polymorphic DNA (RAPD) technique and intergenic 16S-23S ribosomal spacer PCR analysis (ITS 16-23S) were also used. Altogether, these three methods allowed the identification of all of strains tested. However, ribotyping has proven to be more effective than the other methods.

We report here the application of pyrolysis-gas chromatography followed by atomic emission detection (AED) for the characterisation of Corynebacterium amycolatum and related species (i.e., C. striatum, C. minutissimum, C. xerosis and the recently described C. freneyi). This phenotypic method, which analyses the whole chemical composition of bacteria, clearly separates C. amycolatum from other species. Moreover, this C. amycolatum group is subdivided into two distinct subgroups. We cannot differentiate the C. minutissimum strains from those of C. striatum. On the other hand, C. freneyi and C. xerosis are clearly distinct from the other species.

Several methods were used to type 64 clinical isolates of coagulase-negative staphylococci (CNS) derived from hospitals in Morocco. The clinical isolates originated principally from blood cultures and wound sources. These isolates provided the opportunity to substantially compare the proficiency of developing molecular techniques with conventional phenotypic tests for use in the identification of clinical staphylococci. The following molecular methods were examined: Utility ribotyping analysis (Ribotyping); PCR analysis performed with 16S-23S ribosomal-DNA intergenic spacer (ITS-PCR); PCR-based random amplified polymorphic DNA (RAPD). The results obtained by the molecular techniques were contrasted to those of conventional phenotypic tests. Conventional phenotypic tests allowed the outright recognition of the majority of isolates (50/64). These 50 isolates were subdivided into 33 novobiocin-susceptible and 17 novobiocin-resistant strains of CNS. However, 2 other novobiocin-susceptible and 12 other novobiocin-resistant isolates remained unclassified by these tests. There was a good agreement between the conventional phenotypic tests and RAPD for the 33 novobiocin-susceptible isolates. But, the RAPD technique permitted the assignment of the two unidentified novobiocin-susceptible isolates to the Staphylococcus hominis species. A complete correlation was obtained between the three molecular tools for recognition of the 12 novobiocin-resistant isolates that were not identified by phenotypic typing; these were in fact identified as 5 Staphylococcus cohnii and 4 Staphylococcus equorum. Three isolates remained unidentified by all three systems of molecular techniques.

In any health care institution, the most important quality indicator for antisepsis is the existence of systems of reference of use, secondly the knowledge of those systems and then the compliance by the different classes of professionals. These refrentials must be regularly evaluated and updated according to general consensus.

We report here the application of pyrolysis-gas chromatography followed by atomic emission detection (AED) for the characterisation of microorganisms. AED measured the quantity of carbon, sulfur and nitrogen in the molecules separated chromatographically. Twenty-three strains, representing eight Corynebacterium species, were tested in this preliminary study. Co-ordinate principal analysis grouped 11 strains in their respective species group. Most of the other strains appear randomly distributed, perhaps because these strains require additional nutrients. These preliminary results show that the method could be used as a tool for the taxonomic and perhaps the epidemiologic characterisation of bacteria.

The distribution of three subspecies comprising Staphylococcus sciuri was determined for a collection of 30 clinical isolates originating from Morocco, the United Kingdom, and France. The sources of these isolates were principally wounds, skin, and soft tissue infections. At the species level, the isolates were identified according to biochemical characteristics and at the subspecies level by the ribotyping technique. PCR analysis performed with the 16S-23S ribosomal DNA intergenic spacer was less powerful for subspecies differentiation. S. sciuri subsp. sciuri was the most frequent subspecies (21 isolates) found in the collection, whereas S. sciuri subsp. rodentium (seven isolates) and S. sciuri subsp. carnaticus (two isolates) were less common. mecA or a mecA-related gene was detected by PCR and Southern blot in all 30 S. sciuri isolates, supporting the suggestion that S. sciuri species are the natural reservoir of the mecA gene. While the linA/linA' gene coding for lincomycin resistance was present in five isolates, an uncharacterized gene for this resistance was suspected in seventeen other isolates.

Residual chlorhexidine concentrations were measured after application of a single dose on the skin of 22 healthy volunteers. Dosage by high-pressure liquid chromatography in the skin cleansers revealed that the residual concentrations were higher than chlorhexidine MICs for most organisms of the resident skin flora and some responsible for hand-borne infections, even 24 h after application.

Four atypical coagulase-negative staphylococcal (CNS) isolates from clinical sources were compared with Staphylococcus epidermidis strains by ribotyping. The ribotypes of the four strains shared close rDNA restriction profiles with those of the S. epidermidis strains used. The DNA sequence encoding 16S rRNA demonstrated 99.9% homology with S. epidermidis. S1 nuclease experiments showed that these atypical strains formed a homogeneous genomic group. DNA-DNA homologies between the S. epidermidis type strain CCM 2124 and the four CNS isolates ranged from 70 to 89%. The guanine-plus-cytosine content of the deoxyribonucleic acid of the four strains ranged from 31 to 32 mol%.

The mecA gene which confers the oxacillin resistance has been searched by PCR in 290 (124 positives, 166 negatives) coagulase-negative staphylococci (CNS) belonging to twelve species. The results were compared with the oxacillin MIC values obtained by agar dilution (4% NaCl) or by the ATB STAPH method (Api-bioMérieux; 0%, 2%, 5% NaCl) and growth inhibitory diameters obtained by agar diffusion with an oxacillin disk placed at 30 degrees C without NaCl, or at 35 degrees C in presence of 2% or 5% NaCl. Sensitivity of oxacillin resistance detection depends upon the salt concentration and the method used. The optimum concentration is 2%. With this concentration, the Api ATB test appears as the more performant (sensitivity: 89.8%). Search for the mecA gene by PCR represents a very interesting method that detects 96.9% of the oxacillin-resistant CNS strains.

The Polymerase Chain Reaction (PCR) was used to detect the qacA gene which encodes antiseptic resistance in 186 clinical isolates of Staphylococcus aureus. The results were compared with those obtained by MIC determination of 4 antiseptics (benzalkonium chloride, hexamidine, chlorhexidine, acriflavine) and for ethidium bromide. The qacA gene was not detected among the 32 susceptible S. aureus strains, but was found in the 70 (85%) of the 82 S. aureus strains resistant to all 5 antiseptics. The gene was also detected in 70 (45%) of the 154 remaining strains that were resistant to at least one antiseptic.

rRNA gene restriction patterns (ribotyping) were compared with phage typing, serotyping, enterotoxins and exfoliatin production in the analysis of 26 Staphylococcus aureus strains isolated from two different nosocomial outbreaks. Total DNA was cleaved by EcoRI restriction endonuclease. After agarose gel electrophoresis and Southern transfer, the hybridization of the membranes was done with radiolabelled 16S rRNA gene from Bacillus subtilis inserted into a plasmid vector. Six to 13 fragments were visualized. A core of common fragments was discerned for all strains tested. A full correlation between ribotyping and conventional markers was observed in only one of the outbreaks studied. In both outbreaks, ribotyping proved helpful in characterizing otherwise untypable strains.

The pharmacokinetics of ofloxacin was studied in 12 intensive care patients, 6 of whom being under mechanical ventilation. All patients had a creatinine clearance greater than 60 ml/min/1.73 m2 and they were given 3 mg/kg. IV ofloxacin within 30 mn, with a twice daily regimen for 7 days. Pharmacokinetic studies were performed on day 1 (D1) and 7 (D7). Between D1 and D7 a significant increase in T 1/2 beta, AUC and blood levels were observed together with a decrease in total body clearance. Creatinine clearance was not modified between D1 and D7 but ofloxacin renal clearance was considerably altered. Abnormalities in ofloxacin renal tubular secretion may account for the drug cumulation which was observed during repeated administration in intensive care patients.

We studied the surface hydrophobicity of 88 Acinetobacter baumannii strains of clinical origin, using both salt aggregation and adherence to paraxylene tests. Strains were divided into 2 groups: the first included 65 strains isolated from various clinical samples (infected catheters, tracheal and bladder devices); the second included 23 strains isolated from skin obtained from healthy controls. High surface hydrophobicity was observed in 92% of the first group of strains and in only 5% of the second.

After having been widely used in biological research, nucleic probes are now becoming available for the purposes of diagnosis in the clinical microbiology laboratories. Due to various technical improvements, an increasing number of kits will be provided and will allow a rapid and specific diagnosis to be carried out for bacterial agents (ie diarrhea, respiratory infections, sexually transmitted diseases...).

Diffusion of ceftriaxone in renal parenchyma after IV injection (2 g) was studied in six patients with normal renal function, undergoing nephrectomy. Cortex and medulla of the kidney were studied. Blood samples, tissular fragments and urine were studied for drug concentration using HPLC and microbiologic assay 1, 12 and 24 h after injection. Both methods led to similar results. No difference in drug concentration was found between cortex and medulla. Tissular concentration of ceftriaxone remain higher than MIC for most Enterobacteriaceae strains until 24 h.

The guanine-plus-cytosine (G + C) content of different species of Staphylococcus and Micrococcus was determined by high-performance liquid chromatography. Purified bacterial DNA was hydrolysed by nuclease P1. The nucleotides were separated by chromatography and quantified by measurement of the optical density at 260 nm. The G + C content of staphylococci ranged from 31.5 to 37.9 moles %, and that of micrococci from 68.7 to 75.2. Most of our results were comparable to those obtained with the thermal method.

The influence of hemodialysis on the pharmacokinetics of ceftriaxone was studied in 5 patients with chronic renal failure, with a glomerular filtration rate of less than 5 ml/min, and treated by regular hemodialysis. A single dose of 2 g ceftriaxone was administered IV at the end of a hemodialysis, and venous samples were drawn 12 and 24 h thereafter. Two hemodialysis were performed at the 44th (HD1) and the 92nd (HD2) hour, and blood samples were drawn simultaneously on arterial and venous sides of the dialyzer at the onset of HD1 and at the end of HD2. Plasma ceftriaxone concentrations were measured on each sample by both microbiological and chromatographic (HPLC) methods. In these patients, ceftriaxone kinetics are considerably longer than in normal subjects, with an elimination half-life of 16 h, an apparent distribution volume of 800 ml/kg, but without decrease of plasma clearance, except in one patient who had hepatic cytolysis at the time of injection. Plasma concentrations on both sides of dialyzers, or before and after hemodialysis, are not significantly different, and the mean hemodialysis clearance ranges between 26 and 30 ml/min/m2 dialyzer area. According to these data, the dose-interval between successive administrations of ceftriaxone 2 g IV should be 48 h in patients with chronic renal failure, and supplemental doses do not appear necessary after hemodialysis.

A 15 years old boy was admitted to the hospital for high fever, and a four month history of abdominal pain and weight loss. Clinical examination showed painful swelling of the left lumbar region. A retro peritoneal mass was revealed by tomodensitometry. There was a marked biological inflammatory syndrome without bacteriological evidence of infectious disease. Final diagnosis was performed by surgery showing a big abscess. Bacteriological culture of pus was positive for a Haemophilus actinomycetemcomitans.

Three cases of ecchymotic cellulitis in 7 to 11 month old infants are reported. Haemophilus influenzae was isolated from blood in two cases. Ecchymotic cellulitis in infants should, in the first place, evoke an Haemophilus influenzae infection. Bacteremia being frequent in this condition, blood cultures should be performed.

The efficacy of curative and preventive treatment by doxycyclin was studied with the same experimental model as during the study of erythromycin [14]. The infectious aerosol was done with a strain of Legionella pneumophila serogroup 1, Philadelphia (ATCC 33152). Male albinos Dunkin-Hartley guinea-pigs were exposed for 30 min to an aerosol dose of 1, 10 or 100 LD 50 (10(3), 10(4) or 10(5) viable organisms). After preliminary assays to find out the efficient posology, doxycyclin was administered intraperitoneally (IP) 18 h after the infection at a dosage of 60 mg/kg/day for 5 days in the animals infected with 1 LD 50, and 75 mg/kg/day in the animals with 10 and 100 LD 50. The preventive treatment (75 mg/kg/day) administered one day before and the day of the infection was tested with animals infected with 10 LD 50. The guinea-pigs were observed 8 days (weight, rectal temperature) and watched over 3 weeks after the end of treatment; serological and bacteriological tests (cardiac blood, lungs, spleen) were performed in every animal; doxycyclin assays (serum, lungs) during treatment were performed by high performance liquid chromatography (HPLC). The treatment of guinea-pigs infected by 1, 10 and 100 LD 50 gave a survival rate of 100%, 75% and 50% respectively. On the other hand the animals which received preventive treatment were all survivors. Seroconversions with antibody titres from 64 to 512 were observed for all the survivors. Bacteriological tests were all negative. The mean doxycyclin concentrations in serum (microgram/ml) and lung (microgram/g) were 1.66 and 11.42 after 24 h of treatment and 5.71 and 18.93 after 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)

We assessed the role of six beta-lactam antibiotics and of storage time at - 20 degrees C, in inactivation of tobramycin in patients' sera. Several ranges of concentrations were used for the combined antibiotics. The tobramycin concentrations were measured both by microbiological assay (plate diffusion) and an enzyme mediated immunoassay technique (EMIT). Using the bioassay method, the results after 8 days of frozen storage were as follows: carbenicillin and ticarcillin (256 mg/l) induced a 40-50% reduction of tobramycin activity, whereas mezlocillin (256 mg/l) had less effect: a 15-20% reduction. After 15 days at -20 degrees C the results were nearly the same except for azlocillin (256 mg/l), increasing its percentage reduction to 20%. The results obtained by EMIT procedure were significantly better after 8 days of frozen storage: only mezlocillin (5% reduction) and azlocillin (10%) were effective. Nevertheless after 15 days at -20 degrees C, the inactivating action of the pre-cited antibiotics were similar to those obtained at the same time with the bioassay method. Piperacillin and cefsulodin never induced any reduction of tobramycin levels, whatever the time of storage or quantification procedure used. So, our opinion is that patients' sera containing a beta-lactam antibiotic in combination with tobramycin should be assayed immediately upon receipt. Keeping it at -20 degrees C is not sufficient to prevent in vitro tobramycin inactivation.

DNA could not be quickly extracted from members of the genus Actinomyces by the usual methods of lysis. Treatment of 7 different actinomyces cells with lysozyme and achromopeptidase, both 5 mg/g wet cells, for 2 h, followed by SDS (0.2%), proteinase K (5 mg/g wet cells) and EDTA (lmM) for 1 h, lysed the cells. The yield obtained in one day was 337 micrograms per 200 mg of bacterial cells. The treatment was also found to work effectively on strains belonging to Veillonella, Staphylococcus, Fusobacterium and Bifidobacterium genera.

The passive haemagglutination reaction for detecting the Staphylococcus aureus fibrinogen affinity was done in a collaborative study. The test was compared to the major tests used in medical microbiology. The test was as efficient as the protein A or coagulase tests. Its simplicity, rapidity and specificity make it a useful tool for the diagnosis of this species.

Six antibiotics active on the cell-wall were tested against a methicillin resistant Staphylococcus aureus P18 strain at the start of exponential growth rate. The concentration was four-fold the MIC determined in a non-hypersaline liquid medium. The peptidoglycan amino-acid content of the various cell wall samples was determined. An antibiotic-free S. aureus P18 and the methicillin-sensitive Cowan I. S. aureus strain were used a controls. Beta lactam antibiotic treatment, oxacillin and cephalothin, induced an increase of alanine content which suggests that only secondary-transpeptidation did not occur. Similar results were obtained using vancomycin. Amino-acid content was not modified by bacitracin, which is in agreement with the results expected. The results obtained for fosfomycin and cefamandole were not easy to interpret.

The bacteriological characteristics and susceptibility to antimicrobial agents of 108 clinical isolates of Staphylococcus lugdunensis and Staphylococcus schleiferi are described. Fifty out of 108 isolates were considered to be responsible for 16 documented infections, including some severe infections (endocarditis, bacteraemia, osteitis). A number of bacteriological characteristics enabled the identification of these species in the clinical microbiology laboratory: the absence of coagulase and protein A, and the presence of a fibrinogen affinity factor and thermonuclease along with other biochemical characteristics (ornithine and arginine decarboxylases, carbohydrate acidification, novobiocin susceptibility) differentiated these new species from other staphylococci; however, they did not possess virulence markers such as toxins or haemagglutinin, but were haemolytic. In this series, almost all isolates were susceptible to 22 antibiotics and 4 antiseptics representative of the main groups of antimicrobial agents. More information is needed on the ecology and epidemiology of these new opportunistic pathogens.