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Biomedical subjects

D M Shlaes

Publications and source records attributed to D M Shlaes.

At least 19 recordsLinked to original sources
PubMed1992

Mutations altering substrate specificity in OHIO-1, and SHV-1 family beta-lactamase.

The OHIO-1 beta-lactamase does not normally hydrolyse oxyimino-beta-lactam substrates like cefotaxime, ceftriaxone, ceftazidime or aztreonam. We were able to select spontaneous mutants of an OHIO-1-bearing strain of Escherichia coli using the antibiotic substrates listed above by enrichment methods of frequencies of 10(-8)-10(-10) for all antibiotics except ceftazidime (frequency less than 10(-10)). Most mutants with increased resistance to the other beta-lactams were also more resistant to ceftazidime. Mutations identified by DNA sequencing included a Gly238----Ser238 substitution identical with the SHV-2 mutation previously described, cysteine and valine substitutions at the identical site, and a Gly242----Cys242 substitution. The Cys238 and Cys242 mutant enzymes had less affinity for aztreonam than had the other mutant enzymes. Hydrolysis of cefotaxime, but not cephaloridine, by the cysteine-substituted enzymes was inhibited by p-chloromercuribenzoate. The mutant enzymes had, in general, greater affinity for the mechanism-based inhibitors sulbactam, clavulanic acid and tazobactam. These results suggest two non-mutually exclusive hypotheses for the structural role of substitutions in this area of the enzyme. Either potential hydrogen-bond donors, such as serine and cysteine, interact directly with the beta-lactam molecules, or the steric bulk of these substitutions distorts the beta-pleated sheet such that the beta-lactam is held in a position favourable for stable binding and catalysis. Finally, our data raise questions about a strategy relying on oligonucleotide-probe technology to detect such mutations, because of the variety of substitutions that give rise to similar phenotypes.

Amino Acid Sequence↗
PubMed1992

Vancomycin resistance in Enterococcus gallinarum.

The vancomycin resistance expressed by several strains of Enterococcus gallinarum was studied. Resistance was expressed constitutively, as demonstrated by analysis of growth and inhibition of peptidoglycan synthesis. E. gallinarum strains were moderately resistant to vancomycin (MIC, 16 micrograms/ml) but were as susceptible as vancomycin-susceptible enterococci to the glycopeptides, teicoplanin, A35512B, A47934, A4103A, and A41030E and the glycopeptide actaplanins A1, B2, and C1. Vancomycin resistance in E. gallinarum was inhibited by beta-lactam antibiotics at concentrations that saturated penicillin-binding protein 6 (PBP 6), as demonstrated by binding competition experiments. Spontaneous mutants (frequency, 10(-8)) were two- to fourfold more resistant to beta-lactam inhibition of vancomycin resistance than the parent strain. PBP binding competition experiments suggested that PBP 6 in the mutants bound less cefotaxime, while binding of penicillin and cefoxitin was unaffected. Both a bioassay method and high-performance liquid chromatography showed that E. gallinarum membranes have enzymatic activity which modifies a model pentapeptide yielding a product that is thought to be a tetrapeptide. This activity could be a D,D-carboxypeptidase. In both the parent E. gallinarum strain and its derivatives that were resistant to the synergistic drug combination, the activity was inhibited by beta-lactams at concentrations which correlated with those that inhibit vancomycin resistance and those that saturate PBP 6. These results suggest the possibility that PBP 6 may be involved in the vancomycin resistance of E. gallinarum and that the putative D,D-carboxypeptidase activity seen in E. gallinarum membranes may be attributable to PBP 6.

Anti-Bacterial Agents↗
PubMed1991

Enterobacter bacteremia: clinical features and emergence of antibiotic resistance during therapy.

OBJECTIVES: To study the effect of previously administered antibiotics on the antibiotic susceptibility profile of Enterobacter, the factors affecting mortality, and the emergence of antibiotic resistance during therapy for Enterobacter bacteremia. DESIGN: Prospective, observational study of consecutive patients with Enterobacter bacteremia. SETTING: Three university tertiary care centers, one major university-affiliated hospital, and two university-affiliated Veterans Affairs medical centers. PATIENTS: A total of 129 adult patients were studied. MEASUREMENTS: The two main end points were emergence of resistance during antibiotic therapy and death. MAIN RESULTS: Previous administration of third-generation cephalosporins was more likely to be associated with multiresistant Enterobacter isolates in an initial, positive blood culture (22 of 32, 69%) than was administration of antibiotics that did not include a third-generation cephalosporin (14 of 71, 20%; P less than 0.001). Isolation of multiresistant Enterobacter sp. in the initial blood culture was associated with a higher mortality rate (12 of 37, 32%) than was isolation of a more sensitive Enterobacter sp. (14 of 92, 15%; P = 0.03). Emergence of resistance to third-generation cephalosporin therapy (6 of 31, 19%) occurred more often than did emergence of resistance to aminoglycoside (1 of 89, 0.01%; P = 0.001) or other beta-lactam (0 of 50; P = 0.002) therapy. CONCLUSIONS: More judicious use of third-generation cephalosporins may decrease the incidence of nosocomial multiresistant Enterobacter spp., which in turn may result in a lower mortality for Enterobacter bacteremia. When Enterobacter organisms are isolated from blood, it may be prudent to avoid third-generation cephalosporin therapy regardless of in-vitro susceptibility.

Adolescent↗
PubMed1991

Imipenem resistance associated with the loss of a 40 kDa outer membrane protein in Enterobacter aerogenes.

An imipenem-resistant strain, Enterobacter aerogenes EA-Z, was isolated from a blood culture. Outer membrane protein (OMP) profiles indicated the loss of a 40 kDa OMP, decreased expression of 42 and 44 kDa OMPs, and increased expression of a 50 kDa OMP in strain EA-Z when compared with imipenem-susceptible clinical isolates of E. aerogenes. The OMP profile of EA-Z was similar to that of strain EA-SI16, an imipenem-resistant E. aerogenes second-step mutant selected on imipenem-containing media. A single-step imipenem-resistant mutant, EA-SI8, had lost expression of only the 40 kDa OMP. No new beta-lactamase could be detected by isoelectric focusing, and no increase in imipenem hydrolysis was seen when EA-Z was compared with imipenem-sensitive controls, even in the presence of added zinc. These data suggest that the 40 kDa OMP of E. aerogenes might be required for the normal diffusion of imipenem across the outer membrane.

Bacteremia↗
PubMed1991

Synergistic killing of vancomycin-resistant enterococci of classes A, B, and C by combinations of vancomycin, penicillin, and gentamicin.

Using both high and low inocula for time-kill curves, we examined the antibiotic killing of clinical isolates of glycopeptide-resistant enterococci (Enterococcus faecium, E. faecalis, and E. gallinarum) belonging to phenotypic resistance classes A, B, and C. None were resistant to high levels (greater than 500 mg/liter) of gentamicin. Vancomycin-penicillin-gentamicin resulted in 2 or more logs of killing above that of the most effective two-antibiotic combination for all strains except two of three E. gallinarum (VanC) strains and a constitutive mutant of a VanB strain. This strategy may be useful clinically.

Drug Resistance, Microbial↗
PubMed1991

Vancomycin susceptibility and identification of motile enterococci.

Thirty-seven clinical isolates of Enterococcus gallinarum and Enterococcus casseliflavus and three type or reference strains of the species were studied with respect to vancomycin susceptibility and key identification characteristics. With the exception of one clinical isolate of E. casseliflavus (MIC, 4 micrograms/ml), MICs of vancomycin were 8 to 32 micrograms/ml. The type strain of E. gallinarum, NCDO 2313, and five of the clinical isolates had similar penicillin-binding protein profiles and shared 90 to 100% DNA homology. Two isolates, identified as E. gallinarum by conventional tests, were shown to be non-pigment-producing E. casseliflavus on the basis of penicillin-binding protein profile and DNA homology. The type and reference strains of E. casseliflavus, ATCC 25788 and ATCC 25789, were nonmotile in our experiments. However, both shared 65 to 100% DNA homology with each other and with five clinical isolates of E. casseliflavus. These data suggest that the MICs of vancomycin observed for strains of E. gallinarum and E. casseliflavus are higher than those usually associated with other enterococci and may be a common property of these species. Additionally, pigment production and motility may occasionally be misleading criteria for definitive identification of these organisms.

Bacterial Proteins↗
PubMed1990

Comparison of vancomycin-inducible proteins from four strains of Enterococci.

Vancomycin-inducible proteins of 39.5 and 39 kDa from respectively, low-level and high-level resistant Enterococci were compared. Electrophoretic, immunoblot and peptide analysis revealed three types of protein, one in a low-level resistant strain of E. faecium, one in 2 high-level-resistant strains of E. faecium, and one in a high-level resistant strain of E. faecalis. The inducible proteins of E. faecium and E. faecalis, of 39.5 and 39 kDa respectively, which may function in a similar fashion (Al-Obeid et al. (1990) Antimicrob. Agents Chemother. 34, 252-256), are not related immunologically.

Bacterial Proteins↗
PubMed1990

Enterococcal resistance to vancomycin and related cyclic glycopeptide antibiotics.

Enterococci belonging to various species resistant to vancomycin and related cyclic glycopeptide antibiotics have been isolated from hospitalized patients in France, the UK and the USA. All such strains examined display inducible synthesis of a membrane protein associated with resistance. The mechanism by which the membrane protein acts has not been definitively established, but it may block the access of the antibiotic to its peptidoglycan target. That the protein could be a bypass enzyme has not been ruled out. Transfer of glycopeptide resistance by conjugation to either Enterococcus faecium or Enterococcus faecalis and by transformation of Streptococcus sanguis Challis has been reported. The structural and regulatory genes encoding this resistance can be localized on plasmid and, apparently, chromosomal DNA. The plasmids encoding this resistance appear to differ from each other and have variable host ranges, but share at least some DNA sequence homology.

Aminoglycosides↗
PubMed1990

OHIO-1 beta-lactamase is part of the SHV-1 family.

The OHIO-1 beta-lactamase gene was subcloned in a 1.16-kilobase TaqI fragment in the 2.4-kilobase chimeric plasmid pSK04. After directional subcloning into M13, the DNA sequence of this fragment was determined. The results showed an open reading frame of 858 base pairs (bp) encoding a protein of 286 amino acids. The structural gene showed 95, 87, and 60% DNA sequence identity with SHV-1, LEN-1, and TEM-1, respectively, and 93, 85, and 62% predicted amino acid sequence identity, respectively. The 87 bp upstream of the OHIO-1 structural gene had 96% identity with the upstream flanking sequence of SHV-1, including the -35 and -10 consensus sequences and the putative ribosomal binding site. A 223-bp DNA probe derived from a PstI-HaeII fragment in the C-terminal sequence of OHIO-1 had predicted 96, 88, and 61% sequence identity with SHV-1, LEN-1, and TEM-1, respectively. This probe hybridized to SHV-1 and poorly to LEN-1, but not to TEM-1 or a variety of other plasmid-mediated beta-lactamase genes, under stringent conditions. Screening of plasmid DNA derived from 40 ampicillin-resistant clinical isolates by Southern hybridization with the 223-bp probe uncovered no strains encoding OHIO-1. Isoelectric focusing of the same collection did identify two strains producing enzymes resembling SHV-1, however. We have also performed a kinetic comparison of OHIO-1, SHV-1, and TEM-1. OHIO-1 and SHV-1 were indistinguishable from each other but could be distinguished from TEM-1. These data clearly place OHIO-1 within the SHV-1 family of beta-lactamases.

Amino Acid Sequence↗
PubMed1990

Polymicrobial bacteremia in the late 1980s: predictors of outcome and review of the literature.

Although polymicrobial bacteremia has been described in several previous series, there has been no recent study of patients using rigorous statistical analysis. Our objective was to characterize a present-day patient population with polymicrobial bacteremia and to define factors prognostic of survival. Polymicrobial bacteremia accounted for 6% of all positive blood cultures at a university hospital and a Veterans Administration hospital over a 2 1/2 year period in the late 1980s. The majority of these patients were elderly with significant underlying diseases, notably malignancies, and 56% of all episodes were nosocomially acquired. Enterobacteriaceae have remained the most common organisms, though the frequency of gram-positive cocci isolated has increased compared to older studies. Gastrointestinal, genitourinary, and skin and soft-tissue sources were the most common, although the incidence of infections due to central venous catheters appeared to be increasing. The source of 25% of bacteremia was not identified despite newer diagnostic techniques. By univariate analysis, mortality, which was 36% overall, correlated with thrombocytopenia, respiratory failure, disseminated intravascular coagulation, encephalopathy, severity of underlying disease, hemolysis, adult respiratory distress syndrome, use of steroids, renal insufficiency, institution, presence of central lines, and nosocomial acquisition. Using stepwise logistic regression analysis, mortality was predicted by respiratory failure, severity of underlying disease, and hemolysis. We conclude that polymicrobial bacteremia remains an important entity in the present-day hospitalized population, with an increasing frequency of gram-positive organisms and catheter sources, and a large proportion of undiagnosed etiologies.

Humans↗
PubMed1989

Inducible resistance to vancomycin in Enterococcus faecium D366.

Strain D366, a clinical isolate of Enterococcus faecium, is resistant (minimum inhibitory concentration [MIC] 32 mg/L) to vancomycin. When exponential-phase cultures were exposed to half the MIC of vancomycin, a lag of 3-4 h occurred before growth resumed. Cells preexposed to 1/2 MICs of vancomycin did not show any lag. Pregrowth of D366 with vancomycin caused resistance to all glycopeptides tested. Pregrowth in vancomycin resulted in synthesis of a 3.95-kDa cytoplasmic-membrane-associated protein. This protein was correlated with resistance in mutants with high-level resistance, in the presence of NaCl, which inhibited the activity of vancomycin, and when several glycopeptides with varying activities were tested. Vancomycin-grown cells appeared abnormal and lysed at a much slower rate than did normal cells. We conclude that (1) vancomycin resistance in D366 is inducible; (2) resistance is correlated with the synthesis of 39.5-kDa cytoplasmic membrane protein; and (3) this protein play an additional role in the inhibition of normal lytic functions.

Aged↗
PubMed1989

Inducible, transferable resistance to vancomycin in Enterococcus faecium, D399.

Enterococcus faecium D399 was isolated from the blood and peritoneal abscess of a patient with intraabdominal sepsis. The patient had not been treated with vancomycin, but the strain was found to be resistant with a MIC of 1000 mg/l. Resistance was inducible and transferable (probably by conjugation) to JH2-2, and correlated with induction of synthesis of a 39 kDa protein. This mechanism appears to be identical to that previously described for E. faecalis A256, suggesting that dissemination of this form of glycopeptide resistance has already occurred. The resistance phenotype of D399, however, differed somewhat from that found in other enterococcal strains with inducible resistance.

Drug Resistance, Microbial↗
PubMed1989

Infections due to Lancefield group C streptococci.

Our experience with group C streptococcal infection over the past 15 years demonstrates an important and emerging role for this hemolytic organism as an opportunistic and nosocomial pathogen. Significant risk factors in this predominantly male population included chronic cardiopulmonary disease, diabetes, malignancy, and alcoholism. Bacteremia occurred in 74% of cases seen in our series. Nosocomial acquisition of infection was observed in 26%, and infection was frequently polymicrobial in nature with gram-negative enteric bacilli isolated most commonly along with group C streptococci. We observed a broad spectrum of infections including puerperal sepsis, pleuropulmonary infections, skin and soft-tissue infection, central nervous system infection, endocarditis, urinary tract infection, and pharyngeal infections. Several cases of bacteremia of unknown source were observed in neutropenic patients with underlying leukemia. New syndromes of infection due to group C streptococci observed in our series included intra-abdominal abscess, epidural abscess, and dialysis-associated infection. Response to therapy and outcome was related to the underlying disease. While the literature suggests that patients with group C endocarditis respond better to synergistic penicillin-aminoglycoside regimens, patient numbers are too small to draw definite conclusions. The clinical significance of antibiotic tolerant group C streptococci remains uncertain. In patients with serious group C infections including endocarditis, meningitis, septic arthritis, or bacteremia in neutropenic hosts, we advocate the initial use of cell-wall-acting agents in combination with an aminoglycoside.

Abdomen↗
PubMed1989

Escherichia coli susceptible to glycopeptide antibiotics.

Mutants of Escherichia coli susceptible to vancomycin were isolated after mutagenesis with nitrosoguanidine. One such mutant was studied extensively. Multiple regression analysis of the relationship between physical properties of 20 glycopeptides and their in vitro activities against the vancomycin-susceptible mutant revealed a significant correlation with molecular mass (P = 0.007). pI, hydrophobicity, and affinity of the glycopeptide for the pentapeptide target were not as important for activity. This suggested that a block of access of the antibiotic to its target could be the major factor determining activity. Outer membrane proteins of the vancomycin-susceptible mutant, resistant parent, and revertant strains appeared normal. The mutant exhibited increased susceptibility to both erythromycin and fusidic acid which was lost in single-step revertants to vancomycin resistance. Polymyxin B nonapeptide was synergistic with erythromycin and fusidic acid against the parent and revertant but not against the susceptible mutant. Analysis of the susceptibilities of control strains of E. coli and Salmonella typhimurium with known defects in lipopolysaccharide (LPS) synthesis revealed that core LPS mutants (Re chemotype) were phenotypically similar to the E. coli mutant under study. However, the LPS core of the mutant migrated slightly less rapidly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than wild-type or revertant core LPS and did not resemble Re chemotype LPS core obtained from Salmonella rfaC and rfaD mutants. These data suggest that defects in LPS core structure other than loss of heptose moieties may also be important in loss of resistance to large, hydrophilic molecules such as glycopeptides.

Anti-Bacterial Agents↗
PubMed1989

Inducible, transferable resistance to vancomycin in Enterococcus faecalis A256.

A strain of Enterococcus faecalis (A256) was isolated from the urine of a patient with urinary sepsis and was found to exhibit susceptibilities (micrograms per milliliter) to various glycopeptides as follows: vancomycin, 256; teicoplanin, 16; 62208, 512; 62211, 4; and 62476, 16. As judged by growth rates before and after exposure to sub-MICs of glycopeptides, vancomycin and 62476 induced self-resistance, 62208 and 62211 induced slight self-resistance, and teicoplanin did not induce self-resistance. Vancomycin induced cross-resistance to all other glycopeptides tested, as judged both in growth experiments and by direct measurement of inhibition of peptidoglycan synthesis in cells exposed to sub-MICs of vancomycin. Thus, the spectra of activity of the glycopeptides were not correlated with their patterns of induction. There was a correlation between the increased synthesis of a 39-kilodalton (kDa) protein located in the cytoplasmic membrane and the induction of resistance. Protoplasts of A256 were susceptible to inhibition of peptidoglycan synthesis by vancomycin at levels similar to those for susceptible strains. Vancomycin resistance was transferable on filters from the parent strain to E. faecalis JH2-2 at a frequency of about 10(-7), and the 39-kDa protein was also inducible by glycopeptides in these transconjugants. We conclude that A256 is resistant to glycopeptides by virtue of the synthesis of a 39-kDa cytoplasmic membrane protein, that this protein is probably involved in preventing access of the glycopeptides to their peptidoglycan targets, and that this resistance is transferable, probably by conjugation.

Bacterial Outer Membrane Proteins↗