Biomedical subjects
Publications and source records attributed to H Monteil.
Clostridium difficile produces two toxins, an enterotoxin and a cytotoxin. The enterotoxin was purified using fast methods (tangential flow filtration, fast protein liquid chromatography). The purified enterotoxin is composed of two subunits (A1 = 41,500, A2 = 16,000) and its pI is 3.5.
The minimal inhibitory concentrations (MIC) of three acylureidopenicillins (azlocillin, mezlocillin, piperacillin) and three third generation cephalosporins (cefotaxime, cefoperazone, cefsulodin) against 249 randomized strains of Pseudomonas spp. representing 10 species other than Pseudomonas aeruginosa, were determined by the agar dilution method in Mueller-Hinton agar and compared by parametric statistical tests. Fresh clinical and environmental hospital isolates belonged to the following species: P. fluorescens (35), P. putida (37), P. stutzeri (17), P. mendocina (4), P. cepacia (37), P. pickettii (22), P. acidovorans (36), P. diminuta (5), P. paucimobilis (23), P. maltophilia (37). Azlocillin inhibited over 88% of Pseudomonas spp at 64 mg/l or less. This good antipseudomonal activity in vitro was comparable to those of piperacillin and cefotaxime with a minimal inhibitory concentrations 50 value of 8 mg/l. Against these Pseudomonas spp, the ranking order of activity was azlocillin = piperacillin = cefotaxime greater than cefoperazone greater than mezlocillin greater than cefsulodin. Like piperacillin or cefotaxime, azlocillin, was effective against ticarcillin--and aminoglycoside--resistant Pseudomonas spp. Against P. maltophilia and P. cepacia, the two Pseudomonas spp other than P. aeruginosa that are important pathogens in opportunistic infections and in their vast majority are resistant to amino glycosides, azlocillin was significantly active. Only in the case of P. maltophilia, cefoperazone had a lower minimal inhibitory concentrations 50 (8 mg/l vs. 32 mg/l).
The volatile fatty acids produced in culture medium by 357 Pseudomonas strains belonging to eight species were determined quantitatively by GLC. The resultant chromatograms were submitted to discriminant analysis. Stable discriminant functions were computed and included in a computerized identification system which also involved some distinctive volatile fatty acids regarded as two-state qualitative characters (presence or absence characters). Using a test group of 249 strains belonging to the studied species, more than 89% of the identifications made by this system agreed with those made by conventional biochemical methods despite the relatively poor differentiation between P. putida and P. fluorescens. When the individual species within the matrices were weighted with prior probabilities reflecting results given by two simple biochemical tests, 96% of the 249 strains were correctly identified.
Two enzyme-linked immunosorbent assays were developed for detection of staphylococcal exfoliative toxins A and B (ETA and ETB) with a double-antibody sandwich protocol. Antibodies against both toxins were purified by affinity chromatography from sheep antisera raised against purified ETA and ETB. These affinity-purified antibodies were free of detectable amounts of antibodies to other staphylococcal antigens and neutralized the actions of ETA and ETB. Alkaline phosphatase was conjugated to these antibodies. The enzyme-linked immunosorbent assay, which could detect at least 3 ng of ETA and ETB per ml, was used to quantitate the toxins in the culture supernatant fluids of staphylococcal strains. Thus, the kinetics of ETA and ETB synthesis and of ETA and ETB release into the supernatant fluids were determined; other determinations included the roles of carbon dioxide concentration, pH, glucose concentration, temperature, and agitation on the production of ETA and ETB.
Mezlocillin pharmacokinetics were investigated in eighteen patients from a surgical service after a 30 minutes infusion with the purpose, on one hand, to compare this way of administration with a bolus intravenous infusion, and on the other hand to confirm or invalidate capacity-limited dose-dependent pharmacokinetics. Dosages were 2 g (n = 8) and 5 g (n = 10). Mezlocillin levels were measured by high performance liquid chromatography. Calculated data obtained with the 30 minutes infusion are different from those resulting from a bolus infusion. Peak levels, alpha and beta are lower ; T 1/2 beta, areas under the curve, and distribution volumes are higher. Most of the calculated data were not in agreement with a capacity-limited kinetic at the two studied dosages. The two regression-lines (2 g and 5 g) of mezlocillin body clearance versus creatinine clearance superimpose exactly. The 30 minutes infusion, and important hemodynamic variations between our 18 patients may partly explain the differences between our data and those published in the literature.
Clinical and bacteriological efficacy of mezlocillin was evaluated in 41 neonates (including 12 premature babies) with clinical and laboratory evidence of bacterial infection, as shown by elevated C-reactive protein serum concentrations. They received intravenous mezlocillin (80 to 100 mg/kg/dose) every 8 h for 10.4 days. The mean serum concentration (+/- S.E.M.) of mezlocillin in full-term neonates was 214 +/- 19.8 mg/l 1 h after the infusion and 52.0 +/- 9.3 mg/l prior to the next infusion. In premature neonates these mean concentrations were respectively 167 +/- 23.4 mg/l and 40.7 +/- 6.7 mg/l. The efficacy of mezlocillin was documented by the decrease in C-reactive protein serum concentrations and by improvement in clinical condition. Therapy with mezlocillin alone proved to be safe and effective when used for non-nosocomial infections during the neonatal period.
Procedures for the preparation, absorption, and titration of Pseudomonas cepacia O and H rabbit antisera are described. Seven O antigens (O1 to O7) for the slide agglutination test and five H antigens (H1, H3, H5, H6, and H7) for the agglutination and H immobilization tests were determined. Nearly 300 strains of P. cepacia isolated from hospitalized patients (a majority from Strasbourg hospitals) were serotyped. The use of P. cepacia serotyping as an epidemiological tool, especially in outbreak situations, was emphasized. Difficulties in obtaining monospecific H antisera are discussed.
Strictly aerobic Gram negative bacteria are found more and more often in human pathological specimens and in the environment. Amongst these bacteria, Flavobacterium poses problems of bacteriological diagnosis as their precise taxonomy is still recent. In this study, conducted on 321 strains of Flavobacterium from various sources, but essentially from patients in the intensive care unit, the authors define the methods and features of identification and the clinical significance of the species of the Flavobacterium genus. One species is particularly important in medical bacteriology, Flavobacterium meningosepticum which can cause septicaemia and neonatal meningitis which are difficult to treat.
Severe infectious diseases treatment often needs a frequent antimicrobial agent blood levels control. These controls are still performed by microbiological assay procedure. High performance liquid chromatography (HPLC) is now allowing a new kind of assay procedure and improves on speed, specificity and sensitivity. We developed a procedure allowing us to monitor every day by routine, five beta-lactam antibiotics with only one analytical column: benzylpenicillin, ampicillin, cloxacillin, mezlocillin and cefotaxime. A single extraction procedure suitable to the five beta-lactam antibiotics and to various body fluids and the use of three mobile phases, permit us to give a quick answer to the clinicians and thus to consider a rapid adaptation of the doses being administered. In consideration of the specificity, it is possible to control blood levels of each beta-lactam antibiotic even by associated antimicrobial treatment, what is sometimes impossible using the microbiological assay procedure. Cefotaxime is well separated from its active metabolite desacetyl-cefotaxime. The disparity of the levels obtained, for the same posology, essentially by prematures and new-borns, but also in case of massive infusions for endocarditis justify the use of a rapid and specific procedure like high performance liquid chromatography (HPLC).
An outbreak of staphylococcal skin infection in neonates was investigated clinically, bacteriologically and epidemiologically with the following findings: (1) In 8 out of 13 cases, exfoliatin-producing staphylococci were present in the bullae, which is unusual with bullous lesions occurring at other ages; (2) exfoliatin producing staphylococci were present in all children with bullous lesions, as well as in carriers; (3) 39% of the phage II group staphylococci studied produced exfoliatin; (4) purulent lesions due to phage II staphylococci which did not produce exfoliatin were observed. The contaminating agent could be identified in most cases.
The membrane-bound ATPase activity of Bacillus subtilis was inhibited by dicyclohexylcarbodiimide (DCCD). The DCCD-reactive proteolipid of B. subtilis was extracted, from labelled or untreated membranes containing F1 or depleted of F1, with neutral or acidic chloroform/methanol. Purification of the [14C]DCCD-binding proteolipid was attempted by column chromatography on methylated Sephadex G-50 and on DEAE-cellulose. The maximal amount of DCCD which could be bound to the purified proteolipid was found to exceed the amount bound by the purified proteolipid extracted from membranes labelled with the lowest [14C]DCCD concentration required for maximal inhibition of the membrane-bound ATPase activity. The radioactive protein peaks eluted by gel filtration and ion-exchange chromatography were analysed by urea-SDS polyacrylamide slab gel electrophoresis and autoradiography. Radioactivity was incorporated into two components of Mr 18 000 and 6000 when proteolipid was purified by methylated Sephadex. The 6000 polypeptide was always present, whatever the extraction and purification procedures. However, the 18 000 polypeptide was present in largest quantity only when proteolipid was extracted from membranes containing F1 and purified by methylated Sephadex. When proteolipid was purified on DEAE-cellulose this [14C]DCCD binding component of Mr 18 000 was absent.
The authors compared the phenotype characteristics of 100 strains of Pseudomonas cepacia mostly of hospital origin to those of the typical mother strain (ATCC 25416). The morphological, cultural and biochemical characteristics (oxidase characteristics, ONPG, LDC, gelatinase and esculine were generally positive), nutritional characteristics (use of numerous organic compounds) and the antibiotypes (usually resistant to betalactamines, aminosides, polymyxins, tetracycline and furans) are reported, together with the differential diagnostic problems between P. cepacia and P. maltophilia (TTR+ methionine requirement). P. pseudomallei (LDC-, ODC-, ADH+, ONPG-) and Agrobacterium sp (urease+, LDC-, ODC-, absence of exoenzymes and use of citrate and malonate). An original classification in 8 biovars (characteristics used : esculine, nitratase, ONPG) permitted epidemiological marking of the strains and a study of their distribution.
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Six new serotypes (G to L) of Flavobacterium meningosepticum are described herein. Cross-reactions between the 12 serotypes, A to L (including the 6 serotypes A to F of Owen and Lapage, 1974) are described. Sixty-six strains isolated in France (61 from Strasbourg) belong to the following serotypes (with number of strains): A (1), F (4), G (51), H (1), I (1), J (6), K (1) and L (1). Use of meningosepticum serotyping is recommended as an epidemiological tool.
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The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step. The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s20,w of 13,4 and an amino acid composition very similar to bacterial ATPases already studied. After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (alpha) and 57 000 (beta) with different charges. Kinetic studies showed that Ca2+ or Zn2+ are required for ATPase activity, although Mg2+ was uneffective. At optimal Ca2+ concentration, the Mg2+ has an inhibitory effect. The Km for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na+ + K+)-ATPase are studied.
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