scholarly journals Activities of Newer Fluoroquinolones against Streptococcus pneumoniae Clinical Isolates Including Those with Mutations in the gyrA, parC, and parELoci

1999 ◽  
Vol 43 (2) ◽  
pp. 329-334 ◽  
Author(s):  
J. H. Jorgensen ◽  
L. M. Weigel ◽  
M. J. Ferraro ◽  
J. M. Swenson ◽  
F. C. Tenover
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Single Mutation ◽  
Topoisomerase Iv ◽  
In Vitro Susceptibility ◽  
Resistant Strains ◽  
Level Resistance

ABSTRACT Resistance to fluoroquinolone (FQ) antibiotics inStreptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA andgyrB) and topoisomerase IV (parC andparE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, ≥8 μg/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) ofgyrA, gyrB, parC, andparE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 μg/ml) and trovafloxacin (MICs, 0.5 to 1 μg/ml) were associated with either a mutation inparC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA,parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.

Canadian national survey of prevalence of antimicrobial resistance among clinical isolates of Streptococcus pneumoniae. Canadian Bacterial Surveillance Network.

1996 ◽  
Vol 40 (9) ◽  
pp. 2190-2193 ◽  
Author(s):  
A E Simor ◽  
M Louie ◽  
D E Low
Keyword(s):  
Streptococcus Pneumoniae ◽  
Antimicrobial Agents ◽  
Clinical Isolates ◽  
Beta Lactam ◽  
Surveillance Network ◽  
Antibiotic Resistant ◽  
Intermediate Resistance ◽  
Resistant Strains ◽  
High Level ◽  
Level Resistance

The antimicrobial susceptibilities of 1,089 clinical isolates of Streptococcus pneumoniae obtained from 39 laboratories across Canada between October 1994 and August 1995 were determined. A total of 91 isolates (8.4%) demonstrated intermediate resistance (MIC, 0.1 to 1.0 microgram/ml) and 36 (3.3%) had high-level resistance (MIC, > or = 2.0 micrograms/ml) to penicillin. Penicillin-resistant strains were more likely to have been recovered from normally sterile sites (P = 0.005) and to be cross-resistant to several beta-lactam and non-beta-lactam antimicrobial agents (P < 0.05). These results indicate that there has been a recent significant increase in the prevalence of antibiotic-resistant S. pneumoniae in Canada.

scholarly journals Sparfloxacin Resistance in Clinical Isolates ofStreptococcus pneumoniae: Involvement of Multiple Mutations in gyrA and parC Genes

1998 ◽  
Vol 42 (9) ◽  
pp. 2193-2196 ◽  
Author(s):  
Hideki Taba ◽  
Nobuchika Kusano
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Clinical Isolates ◽  
Amino Acid Substitutions ◽  
Sequencing Analysis ◽  
High Level ◽  
Additional Nucleotide ◽  
Level Resistance

ABSTRACT Antimicrobial susceptibility testing revealed among 150 clinical isolates of Streptococcus pneumoniae 4 pneumococcal isolates with resistance to fluoroquinolones (MIC of ciprofloxacin, ≥32 μg/ml; MIC of sparfloxacin, ≥16 μg/ml). Gene amplification and sequencing analysis of gyrA andparC revealed nucleotide changes leading to amino acid substitutions in both GyrA and ParC of all four fluoroquinolone-resistant isolates. In the case of strains 182 and 674 for which sparfloxacin MICs were 16 and 64 μg/ml, respectively, nucleotide changes were detected at codon 81 in gyrA and codon 79 in parC; these changes led to an Ser→Phe substitution in GyrA and an Ser→Phe substitution in ParC. Strains 354 and 252, for which sparfloxacin MICs were 128 μg/ml, revealed multiple mutations in both gyrA and parC. These strains exhibited nucleotide changes at codon 85 leading to a Glu→Lys substitution in GyrA, in addition to Ser-79→Tyr and Lys-137→Asn substitutions in ParC. Moreover, strain 252 showed additional nucleotide changes at codon 93, which led to a Trp→Arg substitution in GyrA. These results suggest that sparfloxacin resistance could be due to the multiple mutations in GyrA and ParC. However, it is possible that other yet unidentified mutations may also be involved in the high-level resistance to fluoroquinolones in S. pneumoniae.

scholarly journals Novel Ser79Leu and Ser81Ile Substitutions in the Quinolone Resistance-Determining Regions of ParC Topoisomerase IV and GyrA DNA Gyrase Subunits from Recent Fluoroquinolone-Resistant Streptococcus pneumoniae Clinical Isolates

2005 ◽  
Vol 49 (6) ◽  
pp. 2479-2486 ◽  
Author(s):  
Nataliya Korzheva ◽  
Todd A. Davies ◽  
Raul Goldschmidt
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
Amino Acid Substitutions ◽  
Topoisomerase Iv ◽  
Isogenic Strains ◽  
New Mutations

ABSTRACT Resistance of Streptococcus pneumoniae to fluoroquinolones is caused predominantly by amino acid substitutions at positions Ser79 of ParC and Ser81 of GyrA to either Phe or Tyr encoded in the quinolone resistance-determining regions of the parC topoisomerase IV and gyrA DNA gyrase genes. Analysis of highly resistant clinical isolates identified novel second-step substitutions, Ser79Leu (ParC) and Ser81Ile (GyrA). To determine contributions of these new mutations to fluoroquinolone resistance either alone or in combination with other Ser79/81 alleles, the substitutions Ser79Leu/Phe/Tyr in ParC and Ser81Ile/Phe/Tyr in GyrA were introduced into the R6 background, resulting in 15 isogenic strains. Their level of fluoroquinolone resistance was determined by susceptibility testing for ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, garenoxacin, and norfloxacin. Leu79 and Ile81 alone as well as 79/81Phe/Tyr substitutions did not contribute significantly to resistance, with fluoroquinolone MICs increasing two- to fourfold compared to wild type for all agents tested. Fluoroquinolone MICs for double transformants ParC Ser79Phe/Tyr/Leu-GyrA Ser81Phe/Tyr were uniformly increased by 8- to 64-fold regardless of pairs of amino acid substitutions. However, combinations including Ile81 conferred two- to fourfold-higher levels of resistance than did combinations including any other Ser81 GyrA substitution, thus demonstrating the differential effects of diverse amino acid substitutions at particular hotspots on fluoroquinolone MICs.

scholarly journals High-Efficiency Generation of Antibiotic-Resistant Strains of Streptococcus pneumoniae by PCR and Transformation

2003 ◽  
Vol 47 (4) ◽  
pp. 1257-1261 ◽  
Author(s):  
Antonio J. Martín-Galiano ◽  
Adela G. de la Campa
Keyword(s):  
Streptococcus Pneumoniae ◽  
High Efficiency ◽  
Spontaneous Mutation ◽  
Single Mutation ◽  
Topoisomerase Iv ◽  
Spontaneous Mutation Rate ◽  
Antibiotic Resistant ◽  
Pcr Products ◽  
Resistant Strains ◽  
Resistant Mutants

ABSTRACT We designed a method by which to generate antibiotic-resistant strains of Streptococcus pneumoniae at frequencies 4 orders of magnitude greater than the spontaneous mutation rate. The method is based on the natural ability of this organism to be genetically transformed with PCR products carrying sequences homologous to its chromosome. The genes encoding the targets of ciprofloxacin (parC, encoding the ParC subunit of DNA topoisomerase IV), rifampin (rpoB, encoding the β subunit of RNA polymerase), and streptomycin (rpsL, encoding the S12 ribosomal protein) from susceptible laboratory strain R6 were amplified by PCR and used to transform the same strain. Resistant mutants were obtained with a frequency of 10−4 to 10−5, depending on the fidelity of the DNA polymerase used for PCR amplifications. Ciprofloxacin-resistant mutants, for which the MICs were four-to eightfold higher than that for R6, carried a single mutation of a residue in the quinolone resistance-determining region: S79 (change to A, F, or Y) or D83 (change to N or V). Rifampin-resistant strains, for which the MICs were at least 133-fold higher than that for R6, contained a single mutation within cluster I of rpoB: S482 (change to P), Q486 (change to L), D489 (change to V), or H499 (change to L or Y). Streptomycin-resistant mutants, for which the MICs were at least 64-fold higher than that for R6, carried a mutation at either K56 (change to I, R, or T) or K101 (change to E). PCR products obtained from the mutants were able to transform R6 to resistance with high efficiency (>104). This method could be used to efficiently obtain resistant mutants for any drug whose target is known.

Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae.

1996 ◽  
Vol 40 (10) ◽  
pp. 2321-2326 ◽  
Author(s):  
X S Pan ◽  
J Ambler ◽  
S Mehtar ◽  
L M Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Low Level ◽  
Drug Concentrations ◽  
Level Resistance

Ciprofloxacin-resistant mutants of Streptococcus pneumoniae 7785 were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains was used to examine the quinolone resistance-determining regions of the GyrA and GyrB proteins of DNA gyrase and the analogous regions of the ParC and ParE subunits of DNA topoisomerase IV. First-step mutants exhibiting low-level resistance had no detectable changes in their topoisomerase quinolone resistance-determining regions, suggesting altered permeation or another novel resistance mechanism. Nine of 10 second-step mutants exhibited an alteration in ParC at Ser-79 to Tyr or Phe or at Ala-84 to Thr. Third- and fourth-step mutants displaying high-level ciprofloxacin resistance were found to have, in addition to the ParC alteration, a change in GyrA at residues equivalent to Escherichia coli GyrA resistance hot spots Ser-83 and Asp-87 or in GyrB at Asp-435 to Asn, equivalent to E. coli Asp-426, part of a highly conserved EGDSA motif in GyrB. No ParE changes were observed. Complementary analysis of two S. pneumoniae clinical isolates displaying low-level resistance to ciprofloxacin revealed a ParC change at Ser-79 to Phe or Arg-95 to Cys but no changes in GyrA, GyrB, or ParE. A highly resistant isolate, in addition to a ParC mutation, had a GyrA alteration at the residue equivalent to E. coli Asp-87. Thus, in both laboratory strains and clinical isolates, ParC mutations preceded those in GyrA, suggesting that topoisomerase IV is a primary topoisomerase target and gyrase is a secondary target for ciprofloxacin in S. pneumoniae.

scholarly journals Amino Acid Substitution in Trichophyton rubrum Squalene Epoxidase Associated with Resistance to Terbinafine

2005 ◽  
Vol 49 (7) ◽  
pp. 2840-2844 ◽  
Author(s):  
Colin S. Osborne ◽  
Ingrid Leitner ◽  
Bertrand Favre ◽  
Neil S. Ryder
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Trichophyton Rubrum ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Squalene Epoxidase ◽  
Reading Frame ◽  
Resistant Phenotype ◽  
Resistant Strains ◽  
Type Sequence

ABSTRACT There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE.

scholarly journals Activities of Clinafloxacin, Gatifloxacin, Gemifloxacin, and Trovafloxacin against Recent Clinical Isolates of Levofloxacin-Resistant Streptococcus pneumoniae

2000 ◽  
Vol 44 (11) ◽  
pp. 2962-2968 ◽  
Author(s):  
J. H. Jorgensen ◽  
L. M. Weigel ◽  
J. M. Swenson ◽  
C. G. Whitney ◽  
M. J. Ferraro ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Clinical Laboratory ◽  
Dna Gyrase ◽  
Test Methods ◽  
Clinical Isolates ◽  
Disk Diffusion ◽  
National Committee ◽  
Sequence Analyses ◽  
Topoisomerase Iv ◽  
Resistant Strains

ABSTRACT The activities of two investigational fluoroquinolones and three fluoroquinolones that are currently marketed were determined for 182 clinical isolates of Streptococcus pneumoniae. The collection included 57 pneumococcal isolates resistant to levofloxacin (MIC ≥ 8 μg/ml) recovered from patients in North America and Europe. All isolates were tested with clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, and trovafloxacin by the National Committee for Clinical Laboratory Standards broth microdilution and disk diffusion susceptibility test methods. Gemifloxacin demonstrated the greatest activity on a per gram basis, followed by clinafloxacin, trovafloxacin, gatifloxacin, and levofloxacin. Scatterplots of the MICs and disk diffusion zone sizes revealed a well-defined separation of levofloxacin-resistant and -susceptible strains when the isolates were tested against clinafloxacin and gatifloxacin. DNA sequence analyses of the quinolone resistance-determining regions of gyrA,gyrB, parC, and parE from 21 of the levofloxacin-resistant strains identified eight different patterns of amino acid changes. Mutations among the four loci had the least effect on the MICs of gemifloxacin and clinafloxacin, while the MICs of gatifloxacin and trovafloxacin increased by up to six doubling dilutions. These data indicate that the newer fluoroquinolones have greater activities than levofloxacin against pneumococci with mutations in the DNA gyrase or topoisomerase IV genes. Depending upon pharmacokinetics and safety, the greater potency of these agents could provide improved clinical efficacy against levofloxacin-resistant pneumococcal strains.

scholarly journals Amino Acid Substitutions in the Cytochrome P-450 Lanosterol 14α-Demethylase (CYP51A1) from Azole-Resistant Candida albicans Clinical Isolates Contribute to Resistance to Azole Antifungal Agents

1998 ◽  
Vol 42 (2) ◽  
pp. 241-253 ◽  
Author(s):  
Dominique Sanglard ◽  
Françoise Ischer ◽  
Luc Koymans ◽  
Jacques Bille
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Antifungal Agents ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Efflux Transporters ◽  
Single Mutation ◽  
Multidrug Efflux ◽  
Cytochrome P 450

ABSTRACT The cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance ofC. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378–2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression inSaccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different clonedCYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.

scholarly journals Sensitivity of human immunodeficiency virus to bicyclam derivatives is influenced by the three-dimensional structure of gp120.

1997 ◽  
Vol 41 (12) ◽  
pp. 2616-2620 ◽  
Author(s):  
K De Vreese ◽  
I Van Nerum ◽  
K Vermeire ◽  
J Anné ◽  
E De Clercq
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Single Amino Acid ◽  
Resistance Pattern ◽  
V3 Loop ◽  
Resistant Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Resistant Strains ◽  
Anti Hiv

The bicyclams are a new class of anti-human immunodeficiency virus (anti-HIV) compounds targeted at viral entry. From marker rescue experiments, it appears that the envelope gp120 glycoprotein plays an important role in the anti-HIV activity of the bicyclams. Bicyclam-resistant strains contain a number of amino acid changes scattered over the V2 to V5 region of gp120. Experiments aimed at estimating the relative importance of particular amino acid changes with regard to the overall resistance pattern are described. The sequences of some partially bicyclam-resistant virus strains, obtained during the resistance development process, were analyzed, and the corresponding 50% effective concentrations were determined. Selected mutations observed in bicyclam-resistant strains were introduced in the wild-type background by site-directed mutagenesis. In addition, some amino acids were back-mutated to their wild-type counterparts in an otherwise JM3100-resistant strain. The sensitivities of these mutant viruses to bicyclams were determined. Construction of chimeric viruses, carrying the V3 loop of JM3100-resistant virus in a wild-type HIV type 1 HXB2 background, enabled us to investigate the importance of the mutations in the V3 loop of JM3100-resistant virus. From the results described in the report, it can be concluded that single amino acid substitutions do not influence the observed resistance to JM3100. Also, the mutations in the V3 loop are not sufficient to engender even a partially resistant phenotype. We postulate that the overall conformation of gp120 determines the degree of sensitivity or resistance of HIV strains to bicyclams.

scholarly journals Intrinsic resistance to terbinafine among human and animal isolates of Trichophyton mentagrophytes related to amino acid substitution in the squalene epoxidase

Infection ◽  
2020 ◽  
Vol 48 (6) ◽  
pp. 889-897 ◽  
Author(s):  
Dominik Łagowski ◽  
Sebastian Gnat ◽  
Aneta Nowakiewicz ◽  
Marcelina Osińska ◽  
Mariusz Dyląg
Keyword(s):  
Amino Acid ◽  
Fungal Infections ◽  
Single Point ◽  
Antifungal Susceptibility ◽  
Point Mutations ◽  
Trichophyton Mentagrophytes ◽  
Single Amino Acid ◽  
Squalene Epoxidase ◽  
Intrinsic Resistance ◽  
Resistant Strains

Abstract Background Dermatomycoses are the most common fungal infections in the world affecting a significant part of the human and animal population. The majority of zoophilic infections in humans are caused by Trichophyton mentagrophytes. Currently, the first-line drug for both oral and topical therapy is terbinafine. However, an increasing number of cases that are difficult to be cured with this drug have been noted in Europe and Asia. Resistance to terbinafine and other allylamines is very rare and usually correlated with point mutations in the squalene epoxidase gene resulting in single amino acid substitutions in the enzyme, which is crucial in the ergosterol synthesis pathway. Purpose Here, we report terbinafine-resistant T. mentagrophytes isolates among which one was an etiological factor of tinea capitis in a man and three were obtained from asymptomatic foxes in Poland. Methods We used the CLSI protocol to determine antifungal susceptibility profiles of naftifine, amphotericin B, griseofulvin, ketoconazole, miconazole, itraconazole, voriconazole, and ciclopirox. Moreover, the squalene epoxidase gene of the terbinafine-resistant strains was sequenced and analysed. Results In the genomes of all four resistant strains exhibiting elevated MICs to terbinafine (16 to 32 µg/ml), single-point mutations leading to Leu393Phe substitution in the squalene epoxidase enzyme were revealed. Among the other tested substances, a MIC50 value of 1 µg/ml was shown only for griseofulvin. Conclusion Finally, our study revealed that the terbinafine resistance phenomenon might not be acquired by exposure to the drug but can be intrinsic. This is evidenced by the description of the terbinafine-resistant strains isolated from the asymptomatic animals.

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