Biomedical subjects
Publications and source records attributed to D Izard.
The species Rahnella aquatilis has been isolated mostly from water, soil, and, in a few cases, from human clinical specimens; little is known about its ecological role. The application of polyacrylamide gel electrophoresis of soluble proteins, DNA-DNA hybridizations and API 20 E systems has shown that Rahnella aquatilis might also be encountered as a contaminant in lager beer breweries.
The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.
A prototype of an expert system for the identification of beta-galactosidase positive Enterobacteriaceae has been developed, for use with the API 20 EC kit. The system is implemented in Prolog on an IBM PC AT with 640 K of central memory and 20 megabytes of secondary memory. Its objectives are to highlight errors that can occur when the kit is in use. It can indicate the presence of new groups or species and give advice or suggest additional tests for the differentiation of the new species from those included in the kit.
Monoclonal antibodies against the alkaline phosphatase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of P. aeruginosa ATCC 10145 and SP20/Ag-14 myeloma cells. The eight stable clones established produced antibodies that reacted by enzyme-linked immunosorbent and indirect immunofluorescence assays with all bacterial strains of P. aeruginosa, including the 17 serotypes and two nontypable strains. Three of the clones cross-reacted only with some Pseudomonas species of the rRNA homology group I defined by N. J. Palleroni (in N. R. Krieg and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, 8th ed., p. 140-218, 1984). The other clones also interacted with other species, including Pseudomonas acidovorans and Xanthomonas maltophilia. Because other species of the genera Aeromonas and Acinetobacter and species of the family Enterobacteriaceae were not detected by these monoclonal antibodies, the antibodies could be used as reagents for routine detection of P. aeruginosa in clinical specimens. Interactions of the antibodies with other Pseudomonas species such as P. fluorescens and P. stutzeri are not important, since these species are susceptible to the same antipseudomonal agents.
A specific monoclonal antibody for Escherichia coli and Shigella sp. alkaline phosphatase was used in an immunocapture assay and allowed identification of E. coli either in culture isolates or directly in clinical specimens. The assay was easy and required only four steps: (i) alkaline phosphatase was released within 10 min by using a gentle lysis procedure, (ii) cell lysates were transferred to antibody-coated tubes for 45 min, (iii) p-nitrophenyl phosphate substrate was added, and (iv) alkaline phosphatase activity was detected in a microsample spectrophotometer at 410 nm. This immunocapture assay was highly specific: only one false-positive reaction was observed with a Klebsiella pneumoniae lysate among the 205 non-E. coli strains tested. The assay was sensitive, detecting 10(7) CFU/ml from culture isolates or 10(5) CFU/ml from urine specimens which had first been grown in phosphate-limiting medium for 2 h. At these bacterial concentrations, the percentages of detected E. coli were high: 91% for blood cultures, 95.4% for culture isolates, and 96.8% for urine specimens.
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Pharmacokinetic values of cefotaxime were measured in 12 cirrhotic male patients (6 without ascites and 6 with ascites) after intravenous injection of a single 2 gram dose of the antibiotic. In patients without ascites elimination of the drug was about the same as in normal subjects or control patients, although clearance was increased. In patients with ascites, the drug elimination half-life was significantly more prolonged (7.5 +/- 3.9 h versus 1.3 +/- 0.4 h, P less than 0.01) and the drug clearance was significantly lower (193.6 +/- 92.4 ml/min versus 475.8 +/- 152.2 ml/min, P less than 0.01) than in the other group. The accumulation of cefotaxime in these patients produced concentration in the ascites fluid that were above the critical therapeutic values for about 20 hours.
Acridine orange was used for staining and counting micro-organisms obtained from 136 skin biopsies performed in burned patients. The number of organisms per gram of tissue was compared to the number of colony-forming-units (CFU) calculated from cultures of the same biopsies. The staining method was positive in 97 per cent of septic samples, and in 25 per cent of these it proved more sensitive than bacterial cultures, with a 100 to 1000-fold greater number of pathogens detected. Acridine orange also demonstrated bacteria in 69 biopsies which remained sterile after culture. In some cases, the same bacterial species was found in other samples taken a few days later.
One hundred and sixty-nine strains of new species of the family Enterobacteriaceae, isolated mainly from the environment, were tested to determine their susceptibilities to 13 antibiotics and 4 antiseptics or disinfectants. All the species were susceptible to aminoglycosides, doxycycline, and trimethoprim but were resistant to chloramphenicol. Susceptibility to beta-lactams varied more among the strains. However, all the strains were cefotaxime susceptible, apart from some Buttiauxella agrestis strains for which MICs were greater than 256 micrograms/ml. The antiseptic MBCs were similar to those published elsewhere for species of the Enterobacteriaceae of clinical origin. No resistance to chlorhexidine was observed. On the other hand, the environmental strains presented a greater resistance to active chlorine than did the reference strains.
A study was performed to compare the Autoscan-4 (MicroScan, Inc., Mahwah, N.J.) with conventional biochemical methods for identifying clinical isolates of the family Enterobacteriaceae. The Autoscan-4 yielded correct identification of 95.4% of the isolates at the species level and 98.4% at the genus level. Only one misidentification was observed. The identification of both common and less-common isolates of Enterobacteriaceae makes this system highly efficient.
A rapid and simple method for preparing chromosomal DNA from gram-negative bacilli is presented. It is based on the alkaline (NaOH 0.03 M) lysis of cell walls. The resulting emulsion is purified by proteinase K (0.625 mg/g of wet wt), SDS, and the deproteinizing agent (chloroform isoamyl alcohol). The purity, molecular nature, and yield of DNA obtained by the present method are compared with those of DNA extracted by Marmur's procedure and a Marmur's modified procedure. We have developed and standardized this original method to isolate double-stranded DNA, free of proteins and RNA contamination and with a significantly higher yield of DNA than the two other methods. This procedure is particularly useful for strains with low growth and can be applied in every field concerned with DNA analysis.
During an 18-month period we identified two cases of septicemia and 24 examples of colonization of humans by Klebsiella trevisanii. Organisms were identified using the API 20EC and API 147 assimilation galleries. Of 147 clinical isolates initially identified as K. oxytoca, 18% were found to be K. trevisanii. Tracheal aspirate was the most common source of the organism. An extensive environmental sampling survey in the rooms of 12 colonized patients revealed a possible reservoir of the organism only once (a face cloth).
The interaction of cefotiam with each of the four aminoglycosides gentamicin, tobramycin, netilmicin and amikacin were studied by the broth microdilution method ("checkerboard" technique) against 36 strains of Enterobacteriaceae chosen for their moderate susceptibility (MIC: 4-32 mg/l) or resistance (MIC: 64-512 mg/l) to cefotiam. A high rate of synergistic combinations was found with all the aminoglycosides: 81% with gentamicin, 76% with amikacin, 67% with tobramycin and netilmicin. The therapeutic value of these interactions appeared excellent.
A broth dilution method was used to measure the minimal inhibitory concentrations of pefloxacin, a new quinolone derivative. Because this new agent could be used in systemic infections, mezlocillin, cefotaxime, ceftazidime and amikacin were used as comparative agents. Pefloxacin was found to be more active than mezlocillin and as active as or more active than cefotaxime, ceftazidime and amikacin. With the exception of Serratia marcescens, only 7% of Enterobacteriaceae strains were resistant to pefloxacin with a MIC greater than 8 micrograms/ml. Pefloxacin was also found to be the best agent against the non-fermenters Staphylococcus aureus and St. epidermidis. Resistance to ampicillin, carbenicillin and gentamicin did not affect the effectiveness of pefloxacin. However, pefloxacin appeared more susceptible to nalidixic acid resistance, with MICs four to sixfold higher.
With automated analysers, bacterial identification and susceptibility testing can be performed in 4-5 hours instead of 12-18 hours with conventional methods. A controlled trial was carried out in the surgical ward of a university hospital to evaluate the clinical repercussions of these rapid methods. The automated analyser reduced delays in the laboratory by about 25%, and optimizing information transfer from laboratory to ward brought the reduction up to 50%. It was found that earlier results of susceptibility testing modified prescriptions and this may be expected to result in a more rational use of antibiotics.
In a bacteriology laboratory where automated and non-automated procedures co-existed during the study period (1 year), patients were randomly assigned to each type of procedure and we observed the physicians behaviour as well as patients well-being in a surgical service using the results from the laboratory. Contrary to our expectations, the reduction in the time delay necessary to obtain information did not alter either the prescribing behaviour of physicians nor the welfare of patients. Besides, the gain in time was significantly lower than expected. We also discuss in detail the meaning and relevance of the results.
The in-vitro activity of ciprofloxacin, a quinolone-carboxylic acid derivative, was compared with those of carbenicillin, azlocillin, cefsulodin, ceftazidime, tobramycin and amikacin against 187 non-fermenters. Only one of the 131 strains of Pseudomonas spp. was not inhibited by 1 mg/l of ciprofloxacin, while these isolates appeared highly resistant to carbenicillin, azlocillin and cefsulodin. Ciprofloxacin was also the best agent against Flavobacterium, Alcaligenes faecalis and Acinetobacter calcoaceticus with MIC90's respectively of 0.5, 4 and 8 mg/l. This new compound appeared bactericidal, and we found a small or no inoculum effect with ciprofloxacin.
The antibacterial activity of ofloxacin was compared to that of nalidixic acid, norfloxacin and against various strains. This new quinolone was about 100 times more active than nalidixic acid, and pipemidic acid and generally 2 to 4 fold more active than norfloxacin and pefloxacin except against Pseudomonas aeruginosa. The increase of resistance to ofloxacin, norfloxacin and pefloxacin seems to plateau, usually at about 16 times the initial MIC. The bactericidal activity of ofloxacin also appeared quickly and more rapidly than that of norfloxacin.