A simple micro-method for time-kill studies amenable to routine laboratory use.
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Humans↗
Biomedical subjects
P Névot
Publications and source records attributed to P Névot.
[Resistance to amoxicillin and amoxicillin-clavulanic acid combination of 231 clinical strains of Escherichia coli, isolated in 1992 at the Cochin Hospital].
Among 231 clinical strains of Escherichia coli tested during may 1992, 89 isolates (38.5%) were resistant to beta-lactams. The resistant strains were principally recovered from urinary and genital specimen from medicine and surgical departments. MICs of beta-lactams were determined alone or combined with clavulanic acid, and beta-lactamases were identified by isoelectric point characterization and by enzymatic inhibition tests. Among the resistant strains, 92.1% were secreting a penicillinase and 6.7% a cephalosporinase. No extended-spectrum beta-lactamase was observed. 85.5% of penicillinases were TEM-1 enzymes, 4.9% SHV-1 beta-lactamase, 1.1% OXA-1 beta-lactamase and 8.5%, 7 strains, were IRT beta-lactamases (formerly called TRI). For 24 clinical E. coli strains, the MICs values were > or = 32 mg/l for amoxicillin plus clavulanic acid. The 7 IRT beta-lactamases showed the highest MICs, 256 to 4096 mg/l. Four of them exhibited a beta-lactamase of pI 5.4 and 3 a beta-lactamase of pI 5.2. The IRT beta-lactamases represent 3% of all the Escherichia coli strains. This frequency is comparable or lower than the values reported by other studies conducted between 1992 and 1994.
Amoxicillin↗
Individualisation of two new genomic groups among American Borrelia burgdorferi sensu lato strains.
We developed a quick typing method for Borrelia burgdorferi sensu lato strains using a fla gene-based PCR assay, followed by dot blot hybridization with non-radioactive species-specific probes. Thirty-six out of 46 strains belonged to one of the four described species (B. burgdorferi sensu stricto n = 11, B. garinii n = 11, B. afzelii n = 9 and B. japonica n = 5) and hybridized with its own species-specific probe. Among the 10 remaining American strains, two new additional genomic groups were identified. This finding was confirmed by direct sequencing of the fla gene-derived amplicons and whole DNA hybridization.
Animals↗
Nucleotide sequences of the genes coding for the TEM-like beta-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2).
Two blaTEM-like genes were characterized that encoded IRT beta-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with beta-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 beta-lactamase resembled the blaTEM-1B gene, and that for IRT-2 resembled blaTEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the blaIRT-1 gene and C to A transversion in the blaIRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to beta-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic pI (pI = 5.2) of these IRT enzymes compared to that of TEM-1 (pI = 5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this beta-lactamase to inhibition by p-chloromercuribenzoate.
Amoxicillin↗
Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to beta-lactamase inhibitors.
The clinical isolate Escherichia coli PEY was highly resistant to amoxycillin, ticarcillin and piperacillin associated to beta-lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam and brobactam but susceptible to cephalosporins, aztreonam and imipenem. The susceptibility to mecillinam indicated that this phenotype was not related to hyperproduction of the TEM-1 beta-lactamase. E. coli PEY produced a new plasmid-mediated inhibitor-resistant beta-lactamase of pI 5.2, which was named IRT-4. The determination of the amino acid sequence (Swiss-Prot accession number, P00810) of the purified protein indicated that IRT-4 differed from TEM-1 by two substitutions: Leu for Met-69 (ABL numbering) and Asp for Asn-276. A Met-69-Leu variant of TEM-1, obtained by site-directed mutagenesis, has been described as resistant to clavulanate. The Asp for Asn-276 substitution has not been reported previously. The side chains of Asp-276 and Arg-244 were expected to interact. Determinations of 50% inhibitory concentrations of beta-lactamase inhibitors and substrate profile of IRT-4 suggested that such an ionic bond was implicated in the alteration of the mechanistic process of TEM-1 beta-lactamase.
Amino Acid Sequence↗
Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of the Borrelia strains used as antigens.
Sera of 52 Lyme borreliosis patients classified according to their clinical features were analysed by Western blot using as antigens Borrelia strains belonging to three recently described genomic species. The antibody response was demonstrated to be homologous within each genospecies. Serum reactivity was studied for each of the type strains Borrelia burgdorferi sensu stricto (strain B31T), Borrelia garinii (strain 20047T) and group VS461. Seven of 15 sera (46.6%) of patients with meningoradiculitis showed preferential reactivity with Borrelia garinii (strain 20047T), all of 8 sera (100%) of patients with acrodermatitis chronica atrophicans showed preferential reactivity with group VS461 (strain VS461) and 8 of 16 sera (50%) of patients with arthritis showed preferential reactivity with Borrelia burgdorferi sensu stricto (strain B31T). The presence of a strong response to OspA and OspB proteins of Borrelia burgdorferi sensu stricto was found only in this last group of patients. These results suggest that there are clinical implications of the recently described modifications in the taxonomy of Borrelia burgdorferi.
Antibodies, Bacterial↗
Clinical isolates of Escherichia coli producing TRI beta-lactamases: novel TEM-enzymes conferring resistance to beta-lactamase inhibitors.
Two different strains of Escherichia coli exhibiting unusual patterns of resistance to beta-lactam antibiotics were isolated from patients at Cochin Hospital. Both isolates showed a low level of resistance to amoxycillin, ticarcillin and ureidopenicillins but were susceptible to cephalosporins, aztreonam and imipenem; beta-lactamase inhibitors potentiated the activities of the beta-lactams to only a limited extent. All resistance characteristics of the strains were transferable by conjugation to E. coli K12. Resistance was shown to be due to beta-lactamases of pI 5.20 and relative molecular masses of 24,000. The hydrolytic and inhibition profiles of these enzymes were similar to each other but differed from those of broad-spectrum beta-lactamases (TEM-1). The rates of hydrolysis (Vmax) of amoxycillin (c. 200%) were higher than that for TEM-1 (84%). Ticarcillin, ureidopenicillins and cephaloridine were hydrolyzed slowly. However, as for TEM-1, no hydrolysis was observed with cefoxitin, third generation cephalosporins, aztreonam and imipenem. The high Km values demonstrated the poor affinity of these enzymes for their substrates. Unlike TEM-1, they were poorly inhibited by beta-lactamase inhibitors. These two enzymes differed from each other as follows: (i) the concentrations of clavulanic acid required for 50% beta-lactamase inhibition were 31 mumol/L for one enzyme (E-SAL) and 9.4 mumol/L for the other (E-GUER); (ii) p-chloromercuribenzoate was a more active inhibitor of E-SAL then E-GUER. The titration curve method and DNA-DNA hybridization studies demonstrated that both enzymes were structurally related to TEM-1. The novel plasmid-encoded enzymes produced by the two isolates of E. coli appeared to be almost identical and to be derived from TEM-enzymes. On the basis of their presumed phylogeny and their biological properties, we propose that these beta-lactamases be given the generic name TRI (TEM Resistant to beta-lactamase Inhibitors).
Anti-Bacterial Agents↗
Susceptibility of Alcaligenes denitrificans subspecies xylosoxydans to beta-lactam antibiotics.
The susceptibility of 56 clinical isolates and two reference strains of Alcaligenes denitrificans subsp. xylosoxydans to beta-lactam agents was tested and related to beta-lactamase activity of the strains. The MICs of 12 beta-lactams determined by an agar dilution method showed that all the strains were sensitive to imipenem and moxalactam. Forty-one cloxacillin-sensitive beta-lactamase producing strains were highly susceptible to azlocillin, piperacillin and ticarcillin, and less susceptible to several cephalosporins (cefamandole, cefoperazone, ceftazidime). The 17 remaining beta-lactamase-producing strains, which were sensitive to clavulanic acid and to a lesser extent cloxacillin, had variable resistance to the penicillins tested and synergy was obtained when these penicillins were combined with clavulanic acid or tazobactam. The choice of agents for treatment of infections with this organism must take into account the susceptibility phenotype of clinical isolates.
Alcaligenes↗
The analysis of five carbenicillin-hydrolysing enzymes by electrophoretic methods.
Five carbenicillin-hydrolysing enzymes (carbenicillinases, or CARB), PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3, CARB-4 and CARB-5, and the beta-lactamase PSE-2 were compared by analysing their isoelectric points (pI), electrophoretic mobilities (mR) and titration curves (pH gradient electrophoresis). The pI determined by isoelectric focusing were 4.3 (CARB-4), 5.3 (PSE-4/CARB-1), 5.7 (PSE-1/CARB-2), 5.75 (CARB-3), 6.1 (PSE-2) and 6.35 (CARB-5). Their mR were estimated by zone electrophoresis as congruent to 26 for PSE-1 (CARB-2), CARB-3 and CARB-5, congruent to 30 for PSE-2, congruent to 33 for PSE-4 (CARB-1) and congruent to 61 for CARB-4. Titration curve analyses indicated that (1) PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5 are closely related variants differing by a few amino acid substitutions; (2) the qualitative titration curve of CARB-4 is different from those of PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5, although their patterns are somewhat similar; and (3) PSE-2 has no structural relationship with any of the other carbenicillin-hydrolysing enzymes or carbenicillinases (CARB) studied. Electrophoretic methods, and in particular titration curve determination combined with other physicochemical and enzymatic data, allowed a rapid comparison of the molecular structures of the beta-lactamases, and hence their classification.
Acinetobacter↗
Analysis of the molecular relatedness of four extended spectrum beta-lactamases (SHV-2, SHV-3, SHV-4 and SHV-5) by comparative protein titration curves.
Six beta-lactamases from Klebsiella pneumoniae, five of which (SHV-1, SHV-2, SHV-3, SHV-4 and SHV-5) were plasmid-encoded and one which (beta 1a GN 11-03) was chromosomally-encoded, were compared by analysis of their isoelectric points (pI), electrophoresis mobilities (MF) and titration curves or pH gradient electrophoresis. Four groups were defined by their pI and MF, namely SHV-1 and SHV-2 (pI = 7.6, MF approximately 14), SHV-3 and beta 1a GN 11-03 (pI = 7.0, MF approximately 20), SHV-4 (pI = 7.8 MF approximately 12) and SHV-5 (pI = 8.2, MF approximately 5). The titration curves of SHV-1 and SHV-2 enzymes on the one hand, and SHV-3 and beta 1a GN 11-03 on the other were completely superimposable for the whole of the pH gradient (3.5-10), indicating strongly similarity. Conservative amino-acid substitutions could account for the differences in the substrate spectra of the purified enzymes. The differences observed between the titration curves of the enzymes SHV-1/SHV-3, SHV-1/beta 1a GN 11-03, SHV-2/SHV-3 and SHV-5/SHV-4 pairs were consistent with the replacement of a basic amino-acid residue in the former enzyme of each pair by an acidic residue in the latter. Similarly, the titration curves of SHV-1/SHV-4 and of SHV-2/SHV-4 pairs may suggest the replacement of an acidic amino-acid in the former beta-lactamases by a neutral amino-acid in the latter of each pair. However, the presence of several self-cancelling or neutral substitutions is also possible. In contrast, when SHV-1 and TEM-1 (pI = 5.4 MF approximately 45) were titrated together, no structural relationship could be inferred.
Chromosomes, Bacterial↗
Novel carbenicillin-hydrolyzing beta-lactamase (CARB-5) from Acinetobacter calcoaceticus var. anitratus.
A strain of Acinetobacter calcoaceticus var. anitratus highly resistant to ticarcillin but susceptible to ticarcillin in combination with clavulanic acid (2 mg/l) was found to produce a constitutive beta-lactamase. This enzyme was periplasmic with a characteristic substrate profile of a carbenicillin-hydrolyzing enzyme. Enzyme inhibition was detected with antiserum (anti-CARB-3), pCMB, cloxacillin, clavulanic acid and sulbactam. This novel enzyme with a molecular mass of 28,000 resembles other plasmid-mediated carbenicillinases (CARB) but differs in its apparent isoelectric point estimated as 6.3 and has been designated CARB-5 on this basis.
Acinetobacter↗
[Titration curves of beta-lactamases using pH gradient electrophoresis].
The molecular relationships of two types of plasmid-mediated beta-lactamases, TEM-1 (R 111), TEM-2 (RP 4) and OXA-1 (RGN 238), OXA-4 (pMG 90) were analysed by combined isoelectrofocusing-electrophoresis. Titration curves of TEM-1 (pI 5.4) and TEM-2 (pI 5.6) together were consistent with the known substitution of a glutamic acid in the former by a lysin in the latter. When OXA-1 (pI 7.4) and OXA-4 (pI 7.45) were titrated, one single mobility curve was obtained reflecting their structural homogeneity. The titration curve technique will be usefull for the study of structure of beta-lactamases.
Electrophoresis, Polyacrylamide Gel↗
[The bacteriostatic and bactericidal effect of amniotic fluid (author's transl)].
The bacteriostatic and bactericidal effect of 100 samples of amniotic fluid (LA) was studied against 5 bacterial species that are responsible for neonatal infections. These results show that there is a bacteriostatic activity in liquor. 52 specimens of liquor amnii (52%) were shown to be active against at least one of the 5 bacterial specimens studied. 32 samples of liquor (32%) were active against Listeria monocytogenes, 21 (21%) against Flavobacterium meningosepticum, 18 (18%) against Escherichia coli, 17 (17%) against group B streptococci and 7 (7%) against Bacteroides fragilis. A simultaneous study of the 6 types of germs show a separate characteristics of this activity. In this way the number of samples of liquor that were active against one or at the same time against 2, 3, 4 or 5 bacteria were respectively 24, 19, 5, 2 and 2. The bacteriostatic effect was more frequently active in those samples of liquor which were studied near term (57.5%) than in the samples studied nearer the beginning of pregnancy (31.5%). All the same, this difference is not statistically significant. There was no difference in the antibacterial activity of samples of liquor from normal and from abnormal pregnancies. The bacterial effect was found only in 12% of samples of liquor, particularly against streptococcus B (8%) and against Listeria monocytogenes (4%). This bactericidal effect was only found after the 31st week of amenorrhoea.
Amniotic Fluid↗
[Determination of antistreptokinases (ASK) by a micromethod].
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Blood Coagulation Tests↗
[Treatment of brucellosis associated with corynebacterium parvum infection in mice with antibiotics (tetracycline, ampicillin, trimethoprim-sulfamethoxazole)].
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Ampicillin↗
[Bronchoscopy and tuberculosis bacilli. Present utility of bronchoscopy in adult phthysiology].
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Adult↗
[Bacterial infections in a department of general medicine].
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Adult↗