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Biomedical subjects

S A Lerner

Publications and source records attributed to S A Lerner.

At least 19 recordsLinked to original sources

Community-acquired pneumonia.

This seminar reviews the aetiology, clinical presentation, approach to diagnosis, and management of immunocompetent adults with community-acquired pneumonia (CAP). Pneumonia is a common clinical entity, particularly among the elderly. A thorough understanding of the epidemiology and microbiology of CAP is essential for appropriate diagnosis and management. Although the microbiology of CAP has remained relatively stable over the last decade, there is new information on the incidence of atypical pathogens, particularly in patients not admitted to hospital, and new information on the incidence of pathogens in cases of severe CAP and in CAP in the elderly. Recent studies have provided new data on risk factors for mortality in CAP, which can assist the clinician in decisions about the need for hospital admission. The emergence of antimicrobial resistance in Streptococcus pneumoniae, the organism responsible for most cases of CAP, has greatly affected the approach to therapy, especially in those patients who are treated empirically. Guidelines for the therapy of CAP have been published by the American Thoracic Society, the British Thoracic Society, and, most recently, the Infectious Diseases Society of America. These guidelines differ in their emphasis on empirical versus pathogenic-specific management.

Community-Acquired Infections

Mechanism of resistance to amikacin and kanamycin in Mycobacterium tuberculosis.

An A1400G mutation of the rrs gene was identified in Mycobacterium tuberculosis (MTB) strain ATCC 35827 and in 13 MTB clinical isolates resistant to amikacin-kanamycin (MICs, >128 microg/ml). High-level cross-resistance may result from such a mutation since MTB has a single copy of the rrs gene. Another mechanism(s) may account for high-level amikacin-kanamycin resistance in two mutants and lower levels of resistance in four clinical isolates, all lacking the A1400G mutation.

Amikacin

Selection and characterization of beta-lactam-beta-lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 beta-lactamase gene.

Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics. This strategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with beta-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM beta-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.

Ampicillin

Trimethoprim/sulfamethoxazole-induced tremor in a patient with AIDS.

OBJECTIVE: To report a case of trimethoprim/sulfamethoxazole (TMP/SMX)-induced tremor responsive to a reduction in dosage. CASE SUMMARY: A 55-year-old white man with AIDS and Pneumocystis carinii pneumonia (PCP) developed a tremor after receiving 5 days of therapy with TMP/SMX 19.4 mg/kg/d (TMP). The tremor resolved completely 3 days after a dosage reduction to TMP/SMX 15.1 mg/kg/d. DISCUSSION: Central nervous system adverse reactions to TMP/SMX have been reported in both the AIDS and non-AIDS populations. To our knowledge, this is the first reported case of TMP/SMX-induced tremor responsive to a reduction in dosage. Pharmacokinetic and clinical data suggest a concentration-dependent etiology for various adverse effects, including tremor. The mechanism of the tremor is unknown; however, toxic metabolites of SMX and disruptions of biogenic amine neurotransmission by TMP have been hypothesized. CONCLUSIONS: TMP/SMX remains the drug of first choice for treating PCP, but it is clearly not well tolerated by patients with AIDS. Concentration-dependent toxicities such as tremor may lead to premature discontinuation of proven, effective TMP/SMX therapy. Using the lower end of the recommended dosing range for TMP/SMX (TMP 15 mg/kg/d) may reduce the incidence of these toxicities while still achieving acceptable TMP concentrations and antimicrobial efficacy.

AIDS-Related Opportunistic Infections

The clinical use of fluoroquinolones for the treatment of mycobacterial diseases.

Mycobacterial diseases often require prolonged therapy with multidrug regimens. Fluoroquinolones have excellent bactericidal activity against many mycobacteria; achieve effective serum, tissue, and intracellular levels following oral administration; and produce few adverse effects. These properties have led to the increasing use of fluoroquinolones for the treatment of mycobacterial infections. We reviewed clinical studies and reports involving the use of fluoroquinolones for mycobacterial diseases. Ofloxacin, ciprofloxacin, sparfloxacin, and pefloxacin exhibit clinical efficacy against mycobacterial diseases, especially tuberculosis and leprosy. Fluoroquinolones have generally been administered in regimens that include other agents. However, when a fluoroquinolone has been found to be the sole active agent in a multidrug regimen, the ready emergence of resistance to fluoroquinolones has been recognized, just as when they have been used as monotherapy. Therefore, to forestall the emergence of resistance to fluoroquinolones during the treatment of mycobacterial diseases, these drugs should always be used in combination with at least one other active agent, and they should be used only when effective alternative drugs are not available.

Anti-Infective Agents

A novel gentamicin resistance gene in Enterococcus.

Enterococcus gallinarum SF9117 is a veterinary isolate for which the MIC of gentamicin is 256 micrograms/ml. Time-kill studies with a combination of ampicillin plus gentamicin failed to show synergism against SF9117. A probe representing aac(6')-aph(2") did not hybridize to DNA from SF9117. A 3.2-kb fragment from plasmid pYN134 of SF9117 was cloned and conferred resistance to gentamicin in Escherichia coli DH5 alpha. Nucleotide sequence analysis revealed the presence of a 918-bp open reading frame whose deduced amino acid sequence had a region with homology to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2"). The gene is designated aph(2")-Ic, and its observed phosphotransferase activity is provisionally designated APH(2")-Ic. An intragenic probe hybridized to the genomic DNA from an Enterococcus faecium isolate from the peritoneal fluid of one patient and to the plasmid DNA of an Enterococcus faecalis isolate from the blood of another patient. An enterococcal isolate containing this novel resistance gene might not be readily detected in clinical laboratories that use gentamicin at 500 or 2,000 micrograms/ml for screening for high-level resistance.

Amino Acid Sequence

Case/control study of the role of splenectomy in chronic lymphocytic leukemia.

PURPOSE: This retrospective analysis was performed to evaluate critically the morbidity and mortality of splenectomy in patients with chronic lymphocytic leukemia and to determine the probability of hematologic response. Further, using a case/control format based on multivariate analysis-derived predictors of survival, we evaluated the influence of splenectomy on survival. PATIENTS AND METHODS: Between 1971 and 1993, 55 patients with chronic lymphocytic leukemia underwent splenectomy. They were compared with 55 fludarabine-treated patients who had been matched for age, serum albumin level, sex, hemoglobin level, Rai stage, number of prior therapies, and time from diagnosis. RESULTS: In the perioperative period, blood-product usage was modest, and common morbidities were limited to minor infections in 18% of the patients and pneumonia/atelectasis in 25%. Perioperative mortality was 9%. Deaths were related to septic complications in all cases and associated with a preoperative performance status > or = 2 (P = .05). The only predictor identified for hemoglobin and neutrophil increments was spleen weight (P < .05). No factors predictive of platelet increment were identified. The early death rate (within 30 days) and overall survival of splenectomy and control patients were not significantly different (P > .2). Among Rai stage IV patients, those who were splenectomized displayed a strong trend for improved overall survival (P = .15 by log-rank test). The 2-year actuarial survival rate of Rai stage IV patients was 51% +/- 9% in the splenectomy group and 28% +/- 9% in the control group. CONCLUSION: Splenectomy can be performed with modest morbidity, mortality, and resource utilization in patients with advanced chronic lymphocytic leukemia and significant cytopenias. The procedure results in major hematologic benefits in most patients, with hemoglobin and neutrophil increments correlated with spleen weight. Overall, the survival of splenectomized patients is equivalent to control patients. Thrombocytopenic patients (< 100 x 10(9)/L) are most likely to obtain hematologic benefit, and potentially enjoy improved survival. These patients would be suitable for a randomized study to establish definitively the role of splenectomy in chronic lymphocytic leukemia.

Adult

Isolation and characterization of a species-specific DNA probe for the detection of Candida krusei.

In an attempt to design species-specific primers for the detection of Candida krusei by polymerase chain reaction, a partial genomic DNA library from Candida krusei was screened for hybridization with radiolabeled genomic probes from a broad variety of fungal and bacterial species and from human. Species-specific candidate DNA inserts were then tested for hybridization with dot blots of DNA from various organisms. One 570-basepair insert from Candida krusei DNA that hybridized under stringent conditions only with DNA from Candida krusei and human was sequenced. It revealed considerable homology with the gene for the mitochondrial inner membrane protease I of Saccharomyces cerevisiae, and the 147 amino acid residues deduced from an open reading frame showed considerable homology with the N-terminal portion of the enzyme from Saccharomyces cerevisiae. From the sequence of the Candida krusei DNA fragment, a pair of 21-base oligonucleotide primers enclosing a 501-basepair sequence was designed for polymerase chain reaction. When these primers were tested with a broad range of genomic DNAs, the expected amplification was obtained only with Candida krusei DNA and not with DNA from any other source, including human. Experiments with DNA from mixed cultures of Candida krusei and other yeasts and bacteria showed that the polymerase chain reaction was specific for Candida krusei and that as few as ten cells could be detected.

Amino Acid Sequence

A comparative study of the broth micro- and macro-dilution techniques for the determination of the in vitro susceptibility of Aspergillus fumigatus.

The effects of inoculum size, medium, temperature, and duration of growth on the in vitro susceptibility testing of Aspergillus fumigatus were investigated using broth micro- and macro-dilution techniques. The minimum inhibitory concentrations (MICs) of ketoconazole, miconazole, itraconazole, fluconazole, and amphotericin B were significantly influenced by the inoculum size, regardless of the techniques used. Two- to four-fold higher MIC values were obtained when the inoculum size was increased 100-fold. The use of peptone yeast extract glucose and RPMI 1640 media provided essentially identical MIC values at 30 and 35 degrees C after incubation for 48 h or longer. A comparison of broth micro- and macro-dilution techniques revealed that, under equivalent conditions, the latter with an inoculum size between 1 x 10(3) and 1 x 10(4) conidia (strain W73355)/mL consistently provided the lowest MICs of fluconazole (256 micrograms/mL), ketoconazole (8 micrograms/mL), miconazole (2 micrograms/mL), itraconazole (0.25 micrograms/mL), and amphotericin B (0.25 micrograms/mL). Using the broth macrodilution technique we screened 24 clinical isolates of A. fumigatus obtained from the Detroit Medical Center in 1994. The MIC values of fluconazole, ketoconazole, miconazole, itraconazole and amphotericin B for all the isolates were 128-256, 8-16, 1-2, 0.25-0.5, and 0.25-1.0 micrograms/mL, respectively, indicating that none of the clinical isolates that we tested shows acquired resistance to the antifungals used.

Antifungal Agents

Characterization of fluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients.

To examine the mechanism of resistance to fluoroquinolones in Mycobacterium tuberculosis, we selected spontaneous fluoroquinolone-resistant mutants from a susceptible strain, H37Rv, and studied the susceptibilities of these mutants and two fluoroquinolone-resistant clinical isolates (A-382, A-564) to various fluoroquinolones and to isoniazid and rifampin. Furthermore, since mutations within the quinolone resistance-determining region of the structural gene encoding the A subunit of DNA gyrase are the most common mechanism of acquired resistance, we amplified this region by PCR and compared the nucleotide sequences of the fluoroquinolone-resistant strains with that of the susceptible strain. Fluoroquinolone-resistant mutants of H37Rv appeared at frequencies of 2 x 10(-6) to 1 x 10(-8). For three mutants selected on ciprofloxacin, ofloxacin, and sparfloxacin, respectively, and the two clinical isolates, MICs of ciprofloxacin and ofloxacin were as high as 16 micrograms/ml, and those of sparfloxacin were 4 to 8 micrograms/ml. They displayed cross-resistance to all fluoroquinolones tested but not to isoniazid or rifampin. Sparfloxacin and FQ-A (PD 127391-0002) were the most potent fluoroquinolones. All of the fluoroquinolone-resistant strains (MICs, > or = 4 micrograms/ml) had mutations in the quinolone resistance-determining region which led to substitution of the Asp residue at position 87 (Asp-87) by Asn or Ala or the substitution of Ala-83 by Val in the A subunit of DNA gyrase. Similar mutations have been noted in other bacterial species and recently in mycobacteria. The broad resistance to fluoroquinolones that arose readily by point mutation in the laboratory and apparently during inadequate therapy, as was the case in the clinical isolates, may ultimately lead to to serious restriction of the use of these drugs in the treatment of tuberculosis.

Aged

Effects of Asp-179 mutations in TEMpUC19 beta-lactamase on susceptibility to beta-lactams.

To examine the effect of disruption of the salt bridge (between Arg-164 and Asp-179 [numbering of Ambler et al. (Biochem J. 267:269-272, 1991)]) that anchors the conserved omega-loop in class A beta-lactamases, we obtained mutant enzymes with each of the 19 other amino acid residues replacing Asp-179 in the TEM beta-lactamase encoded by pUC19 and studied the level of resistance to various beta-lactams conferred by each enzyme. All mutations of Asp-179 compromised the level of resistance to ampicillin, but most of them enhanced resistance to ceftazidime. In contrast, mutations of Asp-179 generally impaired the low levels of resistance to cefepime and aztreonam. One might expect to find clinical isolates with mutant TEM beta-lactamases with replacements of Asp-179 that express an expanded spectrum of resistance to beta-lactams including ceftazidime.

Ampicillin Resistance

Purification and characterization of aminoglycoside 3'-phosphotransferase type IIa and kinetic comparison with a new mutant enzyme.

Aminoglycoside 3'-phosphotransferase [APH(3')s] provide an important means for high-level resistance to neomycin- and kanamycin-type aminoglycoside antibiotics. A four-step purification which affords milligram quantities of homogeneous APH(3') type IIa [APH(3')-IIa] is described. The kinetic parameters for the turnover of five substrates by the enzyme were determined, and the pH dependence and metal activation for catalysis were investigated. All five cysteines in the amino acid sequence of the enzyme exist in their reduced forms; hence, there are no disulfide bonds in the protein. Modification of the cysteine thiols by S-cyanylation showed essentially no effect on the enzymatic activity. A mutant enzyme derived from APH-3'-IIa, which possesses a conservative Glu-182-Asp point mutation and which provides diminished resistance to G418 (R. L. Yenofsky, M. Fine, and J. W. Pellow, Proc. Natl. Acad. Sci. USA 87:3435-3439, 1990), was also purified to homogeneity. Kinetic analysis of this mutant protein indicated an increase of approximately ninefold in the Km for Mg2+ ATP. Insofar as Km may approximate Ks, this finding argues for the involvement of residue 182 in the binding of Mg2+ ATP. Thus, purified APH(3')-IIa and a point mutant derivative enzyme were characterized enzymologically, and the roles of metal cofactors and the five reduced cysteine residues were probed in the wild-type enzyme.

Amino Acids

Reversal of clavulanate resistance conferred by a Ser-244 mutant of TEM-1 beta-lactamase as a result of a second mutation (Arg to Ser at position 164) that enhances activity against ceftazidime.

The mutation of Arg-244 to Ser (Arg-244-->Ser mutation) in the TEM-1 beta-lactamase has been shown to produce resistance to inactivation by clavulanate in the mutant enzyme and resistance to ampicillin plus clavulanate in a strain of Escherichia coli producing this enzyme. The Arg-164-->Ser mutation in the TEM-1 beta-lactamase (TEM-12 enzyme) is known to enhance the activity of the enzyme against ceftazidime, resulting in resistance to the drug in a strain producing the mutant enzyme (D. A. Weber, C. C. Sanders, J. S. Bakken, and J. P. Quinn, J. Infect. Dis. 162:460-465, 1990). The doubly mutated derivative of the TEM-1 enzyme (Ser-164/Ser-244) retains the characteristics of the Ser-164 mutant enzyme, i.e., enhanced activity against ceftazidime and sensitivity to inactivation by clavulanate. It also confers the same phenotype as the Ser-164 mutant enzyme, i.e., resistance to ceftazidime and ampicillin, with reversal of this resistance in the presence of clavulanate. Thus, the Arg-164-->Ser mutation in the TEM-1 beta-lactamase suppresses the effect of the Arg-244-->Ser mutation which, by itself, reduces the sensitivity of the enzyme to inactivation by clavulanate.

Anti-Bacterial Agents

Critical hydrogen bonding by serine 235 for cephalosporinase activity of TEM-1 beta-lactamase.

The role of Ser-235 in the catalytic mechanism of the TEM-1 beta-lactamase has been explored by the study of a mutant enzyme in which Ser-235 has been substituted by alanine (Ala-235 mutant enzyme). A comparative kinetic analysis of both the wild-type and the Ala-235 TEM-1 enzymes revealed little effect of this substitution of residue 235 on the turnover of penicillins but a greater effect on the turnover of cephalosporins. Susceptibility testing of Escherichia coli strains harboring the wild-type TEM-1 beta-lactamase and the Ala-235 mutant enzyme revealed an effect of the mutation similar to that observed in the enzymological studies. The MICs of two representative cephalosporins for the strain containing the mutant enzyme were much lower than those for the isogenic strain bearing the wild-type TEM-1 beta-lactamase. On the other hand, the strain with the mutant enzyme was still highly resistant to penicillins.

Base Sequence

Elucidation of the role of arginine-244 in the turnover processes of class A beta-lactamases.

The highly conserved arginine-244 of beta-lactamases has been postulated to play a role in their initial recognition of substrates, presumably through ion pairing interactions [Moews, P. C., Knox, J. R., Dideberg, O., Charlier, P., & Frère, J. M. (1990) Proteins: Struct., Funct., Genet. 7, 156-171]. However, in the Michaelis enzyme-substrate complex, no direct function has been attributed to this residue. Two mutants with substitutions of this residue in the TEM-1 beta-lactamase (lysine-244 and serine-244) have been prepared to explore whether the guanidinium group of arginine-244 plays a critical role in the turnover processes. The mutant enzymes are effective catalysts for the hydrolysis of both penicillins and cephalosporins, and the lysine mutant enzyme behaves virtually identically to the wild-type beta-lactamase. Comparative kinetic characterization of the serine mutant and wild-type enzymes attributed apparent binding energies of 1.3-2.3 kcal/mol for the penicillins and 0.3-1.0 kcal/mol for the cephalosporins to the transition-state species by arginine-244. Furthermore, it was shown that arginine-244 also contributes equally well to ground-state binding stabilization. These results were interpreted to indicate the involvement of a long hydrogen bond between arginine-244 and the substrate carboxylate, both in the ground and transition states. A reassessed picture for substrate anchoring involving interactions of the substrate carboxylate with the side chains of Ser-130, Ser-235, and Arg-244 is proposed to accommodate these observations.

Arginine

Nephrotoxicity and ototoxicity of aztreonam versus aminoglycoside therapy in seriously ill nonneutropenic patients.

A randomized double-blind clinical trial was done of aztreonam versus aminoglycoside therapy for the empiric treatment of seriously ill nonneutropenic patients suspected of aerobic gram-negative bacterial infection. Each patient was treated for greater than or equal to 72 h with the study drug. Nephrotoxicity, defined by greater than or equal to 50% increase in baseline serum creatinine, occurred in 12 (15%) of 92 patients receiving aminoglycoside therapy and 1 (1%) of 92 patients receiving aztreonam (P less than .004). More severe nephrotoxicity, defined by greater than or equal to 100% increase in baseline serum creatinine, occurred in 6 (6.5%) of 92 patients receiving aminoglycoside therapy and in 1 of 92 receiving aztreonam (P less than .11). Patients with an elevated baseline total bilirubin level were most likely to develop nephrotoxicity. Auditory toxicity occurred in 2 (7%) of 28 evaluatable patients receiving aminoglycoside therapy and in 1 (3%) of 33 receiving aztreonam (P less than .58). One patient, who received aminoglycoside, developed vestibular toxicity. In nonneutropenic patients believed to be at increased risk for renal dysfunction, aztreonam is a less toxic alternative to aminoglycoside therapy for treatment of suspected aerobic gram-negative infection.

Aminoglycosides

Single daily dose therapy with aminoglycosides.

Although aminoglycosides are generally infused every 8 or 12 h, recent data suggest that administration of the total daily dose every 24 h may reduce the risk of oto- and nephrotoxicity and improve efficacy. Aminoglycosides kill bacteria in a dose-dependent manner (i.e. the higher the drug level, the more rapid and complete the bactericidal effect), and exert a post-antibiotic effect (delay in regrowth after the drug concentration falls below its minimum inhibitory concentration for the strain). Moreover, survivors after initial aminoglycoside exposure are temporarily relatively insensitive to the drug, so longer interdose intervals allow recovery of greater sensitivity. Thus, dosing to achieve higher peak drug levels less often would seem desirable. In several animal models of infection, greater efficacy and less oto- and nephrotoxicity have been associated with once daily dosing of aminoglycosides. Several clinical studies have indicated the importance of high peak serum aminoglycoside levels for efficacy. Limited studies of once daily dosing of these drugs in man have suggested that it is at least as favourable as conventional dosing. Nonetheless, despite the obvious advantages of cost and convenience and the theoretical and experimental indication that once daily dosing is advantageous, we need to test this concept in the clinical situation. Large prospective, randomized, double-blind, comparative studies of once daily and conventional dosing regimens in the treatment of various infections in different patient populations are required.

Aminoglycosides