Biomedical subjects
Publications and source records attributed to L Gutmann.
Life-threatening myoclonus developed in a patient following inadvertent intrathecal usage of 60% diatrizoate meglumine. Rhabdomyolysis and myoglobinuria occurred. The patient was successfully treated with midazolam and vecuronium and complete recovery occurred.
Many oral penicillins and cephalosporins are used to treat clinical infections caused by Streptococcus pneumoniae. Therefore, using different beta-lactams as selectors, we estimated the frequencies of one-step mutations leading to resistance. Resistant mutants were obtained from penicillin-susceptible, intermediately resistant, and penicillin resistant strains. For cefixime, cefuroxime, cefpodoxime, cefotaxime, and ceftriaxone, the frequencies of mutation ranged from 10(-6) to 10(-8) when resistant mutants were selected at 2- to 8-fold the MIC, and the MICs increased 2- to 16-fold. For ampicillin, ampicillin-sulbactam, amoxicillin, amoxicillin-clavulanic acid, cefaclor, and loracarbef, the frequencies of mutation were about 10(-7) to 10(-8), and the MICs increased twofold at most. One to three resistance profiles of the resulting mutants were selected for each of the selecting antibiotics. Among those, some showed resistance to the cephalosporins associated with a 2- to 32-fold increase in susceptibility to the penicillins. Competition experiments showed a decreased affinity of PBP2x for cefpodoxime in all mutants. In some mutants that were more susceptible to amoxicillin, a decreased affinity of PBP2x for cefpodoxime was associated with an increased affinity for amoxicillin and a particular substitution of alanine for threonine at position 550 just after the KSG triad. From these results we infer (i) that among the beta-lactams tested the penicillins, cefaclor, and loracarbef selected one-step resistant mutants less frequently and that they achieved a lower level of resistance, and (ii) that mutants with different profiles may have acquired different point mutations in PBP2x.
Induction of vancomycin resistance in Enterococcus faecium D366, which exhibits a VanB-type resistance, as well as its constitutive expression in MT9, a derivative of D366, was associated with penicillin tolerance as shown by decreased lysis and killing of the cells. This phenomenon was linked neither to decreased expression of the different autolysins nor to their decreased lytic activity on the different cell walls. The only change observed was that almost twice the normal amount of D-alanine was attached to the lipoteichoic acid.
The mechanism of high-level fluoroquinolone resistance was studied in strains of Streptococcus pneumoniae, either selected in vitro or isolated from clinical samples. By using DNA from these high-level-resistant strains, low-level-resistant transformants (MIC of pefloxacin, > or = 32 micrograms/ml; MIC of ciprofloxacin, 4 micrograms/ml; MIC of sparfloxacin, 0.50 micrograms/ml) were obtained at high frequencies (ca.10(-2)), while high-level-resistant transformants (MIC of pefloxacin, > or = 64 micrograms/ml; MIC of ciprofloxacin, 16 to 64 micrograms/ml; MIC of sparfloxacin, > or = 8 micrograms/ml) were obtained only at low frequencies (ca.10(-4)). This suggested that mutations in at least two unlinked genes were necessary to obtain high-level resistance. Low-level resistance was associated with ParC mutations (change from Ser to Tyr at position 79 [Ser79Tyr], Ser79Phe, or Asp83Gly). ParC mutations were associated, in high-level-resistant strains and transformants, with alterations in the quinolone resistance-determining region of GyrA (Ser84Tyr, Ser84Phe, and/or Glu88Lys). Low-level resistance was shown to be necessary for expression of the gyrA mutations. No mutation in the region corresponding to the quinolone resistance-determining region of GyrB and no alteration of drug accumulation were found.
Quinolone-resistant Escherichia coli strains were isolated from poultry clinical samples in Saudi Arabia. The poultry flocks had been treated with oxolinic acid or flumequine prophylaxis. The measure of the uptake of fluoroquinolones showed that none of the strains had a reduced accumulation of quinolones. The result of complementation with the wild-type E. coli gyrA gene, which restored fluoroquinolone susceptibility, and the isolation of DNA gyrase from six isolates indicated that the resistant strains had an altered DNA gyrase. The minimum effective dose of ciprofloxacin for inhibition of supercoiling catalyzed by the isolated gyrases varied from 0.085 microgram/ml for a susceptible isolate (MIC < 4 micrograms/ml) up to 96 micrograms/ml for the more resistant one (strain 215, MIC > 64 micrograms/ml). For the same two isolates, the minimum effective doses of sparfloxacin varied from 0.17 up to 380 micrograms/ml. The in vitro selection of spontaneous single-step fluoroquinolone-resistant mutants using ciprofloxacin suggested that the more resistant mutants are likely the result of several mutations. These results also show that, as in human medicine, cross-resistance between older quinolones and fluoroquinolones can exist in veterinary isolates and reiterate the need for the prudent use of these drugs.
A chromosomal gene of Enterobacter cloacae affecting the synthesis of major outer membrane proteins in E. cloacae and Escherichia coli was cloned by using selection for resistance to cefoxitin in E. coli. The presence of the gene, when plasmid-borne, led to a decrease in the amount of porin F in E. cloacae and the amount of OmpF in E. coli and caused 2- to 32-fold increases in the MICs of chloramphenicol, tetracycline, quinolones, and beta-lactam antibiotics. The gene encoded a 33-kDa protein, similar (83% identity) to the protein Rob involved in the initiation of DNA replication in E. coli, which was called RobA(EC1) by analogy. RobA from E. cloacae was found to inhibit ompF expression at the posttranscriptional level via activation of micF, a gene also apparently present in E. cloacae, as detected by PCR. As with its homolog from E. coli, RobA(EC1) is related to the XylS-AraC class of positive transcriptional regulators, along with MarA and SoxS, which also cause a micF-mediated decrease in the level of ampF expression.
The structures of cytoplasmic peptidoglycan precursor and mature peptidoglycan of an isogenic series of Staphylococcus haemolyticus strains expressing increasing levels of resistance to the glycopeptide antibiotics teicoplanin and vancomycin (MICs, 8 to 32 and 4 to 16 microg/ml, respectively) were determined. High-performance liquid chromatography, mass spectrometry, amino acid analysis, digestion by R39 D,D-carboxypeptidase, and N-terminal amino acid sequencing were utilized. UDP-muramyl-tetrapeptide-D-lactate constituted 1.7% of total cytoplasmic peptidoglycan precursors in the most resistant strain. It is not clear if this amount of depsipeptide precursor can account for the levels of resistance achieved by this strain. Detailed structural analysis of mature peptidoglycan, examined for the first time for this species, revealed that the peptidoglycan of these strains, like that of other staphylococci, is highly cross-linked and is composed of a lysine muropeptide acceptor containing a substitution at its epsilon-amino position of a glycine-containing cross bridge to the D-Ala 4 of the donor, with disaccharide-pentapeptide frequently serving as an acceptor for transpeptidation. The predominant cross bridges were found to be COOH-Gly-Gly-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Gly-Gly-NH2. Liquid chromatography-mass spectrometry analysis of the peptidoglycan of resistant strains revealed polymeric muropeptides bearing cross bridges containing an additional serine in place of glycine (probable structures, COOH-Gly-Ser-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Ser-Gly-NH2). Muropeptides bearing an additional serine in their cross bridges are estimated to account for 13.6% of peptidoglycan analyzed from resistant strains of S. haemolyticus. A soluble glycopeptide target (L-Ala-gamma-D-iso-glutamyl-L-Lys-D-Ala-D-Ala) was able to more effectively compete for vancomycin when assayed in the presence of resistant cells than when assayed in the presence of susceptible cells, suggesting that some of the resistance was directed towards the cooperativity of glycopeptide binding to its target. These results are consistent with a hypothesis that alterations at the level of the cross bridge might interfere with the binding of glycopeptide dimers and therefore with the cooperative binding of the antibiotic to its target in situ. Glycopeptide resistance in S. haemolyticus may be multifactorial.
Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in beta-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 micrograms/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k+2/K] values of 17 to 25 M(-1) s(-1)) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 micrograms/ml) and H80721 (MIC, 512 micrograms/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm variants (k+2/K < or = 1.5 M(-1) s(-1)) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.
Two patients with acute onset of profound weakness and loss of muscle bulk during a critical illness had unusual histopathological changes of type II myofibers. Both patients had respiratory failure and prolonged neuromuscular blockade. High-dose steroids were used in only one case and atracurium in the other. Muscle biopsies at days 30 to 32 demonstrated widespread atrophy, basophilic cytoplasm, and vesicular nuclei, features suggestive of regeneration involving virtually all type II myofibers and sparing type I myofibers. This may be another variant or critical illness myopathy or a variation of the pathology during the course of illness.
Abnormalities of peripheral nerve Na+ and K+ channels result in clinical manifestations unrelated to axonal degeneration or demyelination. Na+ channel blockade causes weakness and sensory loss associated with slowed conduction and decreased motor and sensory action potential amplitudes. K+ channel abnormalities result in high frequency repetitive action potentials (e.g., myokymia). Ion channel abnormalities may also play an important role in electrophysiologic changes seen in demyelinating disorders.
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Bacteriocin susceptibilities indicate that during cloacin DF13 uptake the F porin of Enterobacter cloacae plays a similar role to that reported for the OmpF porin of Escherichia coli during colicin A entry. The translocatory activities of these two porins during the bacteriocin uptake can be substituted by the porins D and OmpC, respectively, under conditions not requiring the receptor binding step. Using anti-peptide antibodies, a peptide located in the internal loop L3 of the Escherichia coli OmpF porin was identified in the D and F porins of Enterobacter cloacae. The results demonstrated the existence of a close relationship between porins in terms of both antigenic determinants and bacteriocin susceptibilities.
Six patients with organophosphorus compound intoxications developed an intermediate syndrome (weakness and fasciculations) and obidoxime was given on eight occasions. The efficacy of the acetylcholinesterase (AChE) reactivator was monitored electrophysiologically by neuromuscular transmission studies using single and repetitive nerve stimulation (20 and 50 Hz) and the activity of the serum (butyryl) cholinesterase (ChE). Dramatic electrophysiologic improvement was seen when obidoxime was given early within 12 h in 3 patients, although evidence of AChE inhibition did not subside completely. When administration of obidoxime was delayed 26 h or more after intoxication on five occasions, electrophysiologic improvement was mild or absent. In one case, 66 h after intoxication with oxydemeton-S-methyl, the neuromuscular block worsened, indicating that aging of the AChE had been completed. The electrophysiologic improvement was always accompanied with better motor function but not necessarily with improvement of the overall clinical status. Serum ChE did not predict the oxime effect at the motor endplate. In humans, the efficacy of oximes in AChE reactivation can be determined rapidly using electrophysiologic studies.
Nine patients with organophosphorus (OP) intoxication developing neuromuscular transmission defects were given pancuronium 1, 2, or 4 mg intravenously (IV). Thirteen patient controls with hypoxic encephalopathy received similar dosages. The responses were monitored electrophysiologically using single and repetitive nerve stimulation (20 and 50 Hz). In OP patients, pancuronium did not alter the amplitude of the single CMAP, whereas its repetitive discharges were reduced. Severe neuromuscular blocks were reversed only partially by pancuronium 4 mg. In less severe blocks, 1 and 2 mg resulted in marked improvement. In the patient controls, pancuronium 4 mg induced a severe neuromuscular block but not with 1 and 2 mg. Pancuronium dosages necessary to reverse severe OP-induced neuromuscular blockade produce a neuromuscular block when AChE activity is normal. Low dosages have little effect on normal neuromuscular transmission, but improve the block to a mild degree and may be useful as part of treatment in OP intoxications.
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To identify risk factors for and prognostic indicators of the nosocomial acquisition of multiresistant Acinetobacter baumannii in an intensive care unit, we prospectively studied 40 patients: 13 who were infected with this organism and 27 who were colonized. Isolates were identified by pulsed-field gel electrophoresis; the infected/colonized patients were compared with 348 noninfected, noncolonized patients by logistic regression analysis and with matched historical controls in a cohort study. The severity of illness (evaluated by the APACHE II score; P < .05) and previous infection (P < .001) were retained as independent risk factors for acquiring A. baumannii. Logistic regression analysis selected a high APACHE II score (P < .01) and the acquisition of A. baumannii (P < .01) as factors independently associated with death. The acquisition of A. baumannii was associated not only with high mortality but also with a length of stay on the intensive care unit in excess of that due to the underlying disease alone; specifically, the attributable mortality was 25%, with a risk ratio for death of 2.0 (95% confidence interval, 1.11-3.62), and the duration of stay for infected/colonized patients was 10.3 days longer than that for controls (P < .001).
A synergistic bactericidal effect between penicillin, vancomycin, and gentamicin has been described against enterococci highly resistant to beta-lactams and glycopeptides. Since such a synergy was also observed in vitro between ceftriaxone, teicoplanin, and gentamicin against Enterococcus faecium 70/90, the efficacy of different combinations including penicillin or ceftriaxone, vancomycin or teicoplanin, and gentamicin was compared in vivo in experimental endocarditis. In vitro, all four triple combinations provided a bactericidal effect. In rabbits, after a 5-day treatment, the ceftriaxone-vancomycin-gentamicin combination was the most effective, both in reducing the total bacterial titers and in eradicating the subpopulation resistant to the synergy. Compared with the 5-day regimen, 10- and 20-day regimens of ceftriaxone-vancomycin-gentamicin, each followed by a 3-day antibiotic-free period, increased the number of sterilized animals but failed to avoid the emergence of resistant bacteria, which occurred in 10%-20% of animals.