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E K Manavathu

Publications and source records attributed to E K Manavathu.

At least 37 records · Page 2Linked to original sources
PubMed1998

In-vitro isolation and antifungal susceptibility of amphotericin B-resistant mutants of Aspergillus fumigatus.

Aspergillus fumigatus mutants resistant to amphotericin B were selected in the laboratory following UV irradiation. A total of 18 colonies (frequency 1.8 x 10(-7)) that grew in the presence of amphotericin B (8 mg/L and 16 mg/L) on peptone yeast extract glucose agar were tested for their susceptibility to amphotericin B, nystatin, azoles, and the echinocandin L-743872. Ten of the 18 isolates showed an eight-fold rise in amphotericin B MIC (4 mg/L) compared with the susceptible parent whereas the remaining isolates showed a 16 to 32-fold rise in amphotericin B MIC (8-16 mg/L). Subculturing of three representatives from each of the groups that had MIC values of 4 mg/L and 8-16 mg/L for six cycles revealed that the resistance trait was stably expressed. All amphotericin B-resistant isolates showed a significant level of cross-resistance to nystatin but not to azoles and L-743872. Kill-curve studies with amphotericin B revealed that the killing of the resistant isolates was significantly less than that of the susceptible parent strain. These results show that amphotericin B-resistant mutants of A. fumigatus can be isolated in the laboratory following a single-step UV-induced mutagenesis and suggest that similar mechanisms could operate in nature for the emergence of resistance in clinical isolates.

Amphotericin B↗
PubMed1998

In-vitro and in-vivo susceptibility of Aspergillus fumigatus to a novel conjugated styryl ketone.

We investigated the in-vitro and in-vivo susceptibility of Aspergillus fumigatus to the novel conjugated styryl ketone NC1175 and the results were compared with those obtained for amphotericin B and itraconazole. All 20 clinical isolates of A. fumigatus examined were susceptible to NC1175 (MIC = 5.54 +/- 2.48 mg/L; range 2.92-11.68 mg/L), and the minimum lethal concentration (MLC) was only twice the MIC, suggesting that NC1175 is fungicidal. The mean MIC values of amphotericin B (1.22 +/- 0.58 mg/L; range 0.5-4 mg/L) and itraconazole (0.37 +/- 0.11 mg/L; range 0.125-0.5 mg/L) were approximately nine- and 22-fold, respectively, lower than that of NC1175. Both amphotericin B-resistant (n = 18) and itraconazole-resistant (n = 28) isolates of A. fumigatus were as susceptible to NC1175 as amphotericin B-, and itraconazole-susceptible isolates. Kill curve experiments revealed that NC1175 at 23.35 mg/L (approximately four times the MIC) killed > or = 99% of conidia within 24 h of exposure to the drug. The in-vivo susceptibility of A. fumigatus to NC1175 was investigated using a murine pulmonary aspergillosis model. Treatment of infected mice with amphotericin B or NC1175 did not result in significant improvement of the mean survival (amphotericin B, 7.05 +/- 0.07 days; NC1175, 6.65 +/- 1.25 days) of the animals compared with that of the placebo group (7.21 +/- 1.20 days). However, semiquantitative organ culture revealed that clearance of A. fumigatus occurred in 16.6%, 50% and 66.6% of the mice treated with placebo, NC1175 and amphotericin B, respectively (P value for the control and the treated groups <0.01). These results suggest that NC1175 has in-vivo and in-vitro activity against A. fumigatus and can be used as a prototypic molecule for further development as an antifungal agent.

Amphotericin B↗
PubMed1998

Organism-dependent fungicidal activities of azoles.

We investigated the antifungal activities of itraconazole and voriconazole on Aspergillus species by time kill studies, and the results were compared with those obtained for Candida species. Exposure of Aspergillus fumigatus conidia to varying concentrations (1.25 to 10 microg/ml) of itraconazole and voriconazole resulted in cellular death; the cytocidal effect was time and concentration dependent. In contrast, no killing of Candida albicans occurred in the presence of itraconazole and voriconazole at concentrations as high as 10 microg/ml, although candidal growth was inhibited compared to the drug-free control. Amphotericin B (1.25 to 10 microg/ml), on the other hand, killed both A. fumigatus and C. albicans. Similar results were obtained for non-A. fumigatus aspergilli and non-C. albicans Candida species. These observations indicate that both itraconazole and voriconazole are cytocidal agents for Aspergillus species but not for Candida species, suggesting that azoles possess organism-dependent fungicidal activities.

Amphotericin B↗
PubMed1998

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↗
PubMed1998

In vitro antifungal activity of some Mannich bases of conjugated styryl ketones.

Four Mannich bases of some conjugated styryl ketones IIa-IId were examined for antifungal activity. These compounds were designed as thiol-alkylators and had two centers for attack by cellular thiols. The most potent compounds IIa and IIb possessed hydrophobic, electron-attracting substituents in the aryl rings and in general had minimum inhibitory concentration (MIC) values of 0.2-25 microM against a variety of fungi. None of the four compounds inhibited the growth of a number of bacteria (MIC > 100 microM). The minimum fungicidal concentration (MFC) values for IIa and IIb were generally either similar or twofold higher than the MIC figures for fungi. Compound IIa demonstrated rapid, concentration-dependent inhibition of the growth of Candida albicans B311. The toxicity of IIa to normal human cells was much lower than the concentrations of this compound required to inhibit fungal growth. In summary, this study of four prototypic molecules has revealed that this class of compounds may have potential for further development as candidate antifungal agents.

Antifungal Agents↗
PubMed1997

In vitro antifungal activity of CAN-296: a naturally occurring complex carbohydrate.

The in vitro activity of a naturally occurring complex carbohydrate, CAN-296, was evaluated by testing 132 clinical and ATCC isolates of yeast and Aspergillus fumigatus, many of which were azole-resistant. The in vitro susceptibility tests were performed by standardized broth micro- and macrodilution methods and results were compared with those obtained for amphotericin B, fluconazole, ketoconazole, flucytosine and the pneumocandin L-733,560. All tested Candida species showed highly uniform susceptibility to CAN-296 at concentrations of 0.078 to 0.312 microgram/ml; non-albicans Candida were as susceptible to CAN-296 as the Candida albicans strains. Multi-azole-resistant Candida species were highly sensitive to CAN-296. Minimum inhibitory concentration measurements did not differ from minimum lethal concentrations by more than two-fold for all tested Candida species. Aspergillus fumigatus, on the other hand, showed only moderate susceptibility to CAN-296. The kinetics of the anti-Candida activity of CAN-296 was investigated by kill-curve experiments using C. albicans and C. glabrata and the results were compared with those obtain for amphotericin B. CAN-296 was found to be rapidly fungicidal in concentrations ranging from 4-16 fold the mean MIC value. The broad spectrum of anti-Candida activity together with the rapid fungicidal effect make this complex carbohydrate a promising agent for clinical use.

Antifungal Agents↗
PubMed1997

Antifungal activity of conjugated styryl ketones.

The susceptibility of Aspergillus fumigatus to a series of alpha, beta-unsaturated styryl ketones known to be thiol-alkylators was examined, and the results were compared with those obtained for Candida albicans. Among 13 compounds used in our study, one (designated NC1110) inhibited the growth of A. fumigatus completely at low concentrations (minimum inhibitory concentration = 32 microM). Structure-activity analysis of these compounds indicated that the electron attracting property as well as the overall hydrophobicity of the compounds are important parameters for their antifungal activity. These preliminary results suggest that further modification of these molecules to enhance their hydrophobicity and the electron attracting property may result in more active compounds with improved antifungal activity.

Antifungal Agents↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1995

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↗
PubMed1994

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↗
PubMed1994

Activity of some Mannich bases of conjugated styryl ketones against Candida albicans.

A number of Mannich bases of conjugated styryl ketones displayed activity against Candida albicans strains 3153A and B311. Antifungal potency was influenced by the relative hydrophobicities of the molecules and on occasions by the electronic nature of the aryl substituents. The compounds inhibited one or more of the following enzymes in the glutathione metabolic pathway namely glutathione S-transferases, glutathione reductase, gamma-glutamyl transpeptidase and glutathione peroxidase. Nearly half of the compounds examined on the yeast-to-mycelium transition in C. albicans 3153A prevented this conversion from occurring.

Antifungal Agents↗
PubMed1993

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↗
PubMed1993

Effect of mutational alteration of Asn-128 in the putative GTP-binding domain of tetracycline resistance determinant Tet(O) from Campylobacter jejuni.

The deduced amino acid sequence of Campylobacter jejuni Tet(O), cloned in Escherichia coli, has shown that it contains the five highly conserved sequences of the GTP-binding domain found in other GTPases. Asn-128 belongs to the G4 motif of such a domain and is involved in hydrogen bonding with the guanine ring of the nucleotide. Substitution of Asn-128 by 11 other amino acids resulted in a decrease in tetracycline resistance, indicating that tetracycline resistance conferred by Tet(O) is related to GTP binding. The effect of the mutations on the GTP-binding domain is discussed with the EF-Tu-GDP complex as a model.

Amino Acid Sequence↗
PubMed1993

Chromosomal localization of phosphoglycerate kinase (PGK) gene in Candida albicans using a heterologous probe.

The glycolytic enzyme phosphoglycerate kinase (PGK) is highly conserved throughout the evolutionary ladder. Using a previously cloned Saccharomyces cerevisiae PGK gene as hybridization probe, chromosomal localization and strain-specific variation of PGK gene in C. albicans have been examined. Separation of chromosome-size DNA fragments by pulse field gel electrophoresis followed by DNA- DNA hybridization analysis revealed that the PGK is encoded by a single copy gene located on the large chromosome of the commonly used laboratory strain ATCC 32354, as well as of several clinical isolates of C. albicans. Restriction fragment length analysis indicated that the PGK gene locus is highly polymorphic, and this strain-specific variation could be exploited to study intra-specific relatedness of clinical isolates.

Candida albicans↗
PubMed1992

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↗
PubMed1991

Isolation and characterization of a species-specific DNA probe for Candida albicans.

As a model for the isolation of species-specific sequences of DNA, we isolated and characterized a species-specific DNA fragment from the Candida albicans genome. We used a series of differential colony hybridization experiments, in which a C. albicans genomic library was hybridized with genomic DNA probes from related organisms to minimize the number of potentially specific C. albicans DNA fragments to be tested. Six clones were tested by dot blot analysis, and one of them, a 1348 bp EcoRI DNA fragment, was confirmed as specific for C. albicans. This species-specific fragment could be utilized as a DNA probe for rapid, sensitive, and specific diagnosis of C. albicans DNA in clinical specimens.

Base Sequence↗