scholarly journals Mutations in the dihydrofolate reductase gene of trimethoprim-resistant isolates of Streptococcus pneumoniae.

1997 ◽  
Vol 41 (11) ◽  
pp. 2406-2413 ◽  
Author(s):  
P V Adrian ◽  
K P Klugman
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Correlation Coefficient ◽  
Dihydrofolate Reductase ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Chromosomal Dna ◽  
Reductase Gene ◽  
Genes Encoding ◽  
Dihydrofolate Reductases

Streptococcus pneumoniae isolates resistant to several antimicrobial agent classes including trimethoprim-sulfamethoxazole have been reported with increasing frequency throughout the world. The MICs of trimethoprim, sulfamethoxazole, and trimethoprim-sulfamethoxazole (1:19) for 259 clinical isolates from South Africa were determined, and 166 of these 259 (64%) isolates were resistant to trimethoprim-sulfamethoxazole (MICs > or =20 mg/liter). Trimethoprim resistance was found to be more strongly correlated with trimethoprim-sulfamethoxazole resistance (correlation coefficient, 0.744) than was sulfamethoxazole resistance (correlation coefficient, 0.441). The dihydrofolate reductase genes from 11 trimethoprim-resistant (MICs, 64 to 512 microg/ml) clinical isolates of Streptococcus pneumoniae were amplified by PCR, and the nucleotide sequences were determined. Two main groups of mutations to the dihydrofolate reductase gene were found. Both groups shared six amino acid changes (Glu20-Asp, Pro70-Ser, Gln81-His, Asp92-Ala, Ile100-Leu, and Leu135-Phe). The first group included two extra changes (Lys60-Gln and Pro111-Ser), and the second group was characterized by six additional amino acid changes (Glu14-Asp, Ile74-Leu, Gln91-His, Glu94-Asp, Phe147-Ser, and Ala149-Thr). Chromosomal DNA from resistant isolates and cloned PCR products of the genes encoding resistant dihydrofolate reductases were capable of transforming a susceptible strain of S. pneumoniae to trimethoprim resistance. The inhibitor profiles of recombinant dihydrofolate reductase from resistant and susceptible isolates revealed that the dihydrofolate reductase from trimethoprim-resistant isolates was 50-fold more resistant (50% inhibitory doses [ID50s], 3.9 to 7.3 microM) than that from susceptible strains (ID50s, 0.15 microM). Site-directed mutagenesis experiments revealed that one mutation, Ile100-Leu, resulted in a 50-fold increase in the ID50 of trimethoprim. The resistant dihydrofolate reductases were characterized by highly conserved redundant changes in the nucleotide sequence, suggesting that the genes encoding resistant dihydrofolate reductases may have evolved as a result of inter- or intraspecies recombination by transformation.

scholarly journals Amino Acid Substitutions in Mosaic Penicillin-Binding Protein 2 Associated with Reduced Susceptibility to Cefixime in Clinical Isolates of Neisseria gonorrhoeae

2006 ◽  
Vol 50 (11) ◽  
pp. 3638-3645 ◽  
Author(s):  
Sho Takahata ◽  
Nami Senju ◽  
Yumi Osaki ◽  
Takuji Yoshida ◽  
Takashi Ida
Keyword(s):  
Amino Acid ◽  
Neisseria Gonorrhoeae ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Complete Sequence ◽  
Clinical Isolates ◽  
Amino Acid Substitutions ◽  
Penicillin Binding Protein ◽  
Fourfold Increase ◽  
Genes Encoding

ABSTRACT The molecular mechanisms of reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae, particularly amino acid substitutions in mosaic penicillin-binding protein 2 (PBP2), were examined. The complete sequence of ponA, penA, and por genes, encoding, respectively, PBP1, PBP2, and porin, were determined for 58 strains isolated in 2002 from Japan. Replacement of leucine 421 by proline in PBP1 and the mosaic-like structure of PBP2 were detected in 48 strains (82.8%) and 28 strains (48.3%), respectively. The presence of mosaic PBP2 was the main cause of the elevated cefixime MIC (4- to 64-fold). In order to identify the mutations responsible for the reduced susceptibility to cefixime in isolates with mosaic PBP2, penA genes with various mutations were transferred to a susceptible strain by genetic transformation. The susceptibility of partial recombinants and site-directed mutants revealed that the replacement of glycine 545 by serine (G545S) was the primary mutation, which led to a two- to fourfold increase in resistance to cephems. Replacement of isoleucine 312 by methionine (I312M) and valine 316 by threonine (V316T), in the presence of the G545S mutation, reduced susceptibility to cefixime, ceftibuten, and cefpodoxime by an additional fourfold. Therefore, three mutations (G545S, I312M, and V316T) in mosaic PBP2 were identified as the amino acid substitutions responsible for reduced susceptibility to cefixime in N. gonorrhoeae.

scholarly journals Prevalence, Characteristics, and Molecular Epidemiology of Macrolide and Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae at Five Tertiary-Care Hospitals in Korea

2007 ◽  
Vol 51 (7) ◽  
pp. 2625-2627 ◽  
Author(s):  
Jeong Hwan Shin ◽  
Hee Jung Jung ◽  
Hye Ran Kim ◽  
Joseph Jeong ◽  
Seok Hoon Jeong ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Molecular Epidemiology ◽  
Tertiary Care ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
New Mutation ◽  
Tertiary Care Hospitals

ABSTRACT The genes erm(B), mef(A), and both erm(B) and mef(A) were identified in 42.6, 10.1, and 47.3%, respectively, of the erythromycin-resistant Streptococcus pneumoniae isolates. Of the strains, 3.8% were nonsusceptible to levofloxacin and had 1 to 6 amino acid changes in the quinolone resistance-determining region, including a new mutation, Asn94Ser, in the product of parC. Levofloxacin with reserpine was highly specific for efflux screening.

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 Amino Acid Repetitions in the Dihydropteroate Synthase of Streptococcus pneumoniae Lead to Sulfonamide Resistance with Limited Effects on SubstrateKm

2001 ◽  
Vol 45 (3) ◽  
pp. 805-809 ◽  
Author(s):  
Ylva Haasum ◽  
Katrin Ström ◽  
Rahma Wehelie ◽  
Vicki Luna ◽  
Marilyn C. Roberts ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Washington State ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Directed Mutagenesis ◽  
Aminobenzoic Acid ◽  
Kinetic Measurements ◽  
Dihydropteroate Synthase ◽  
Gene Coding ◽  
Sulfonamide Resistance

ABSTRACT Sulfonamide resistance in Streptococcus pneumoniae is due to changes in the chromosomal folP (sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniaeisolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile66 and Glu67 in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folP gene, identical to the sul-d mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in Ki for sulfathiazole but changed the Km for p-aminobenzoic acid only 2.5-fold and did not significantly change theKm for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser61 had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.

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 Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction of l-Leucine to Isocaproate by Clostridium difficile

2006 ◽  
Vol 72 (9) ◽  
pp. 6062-6069 ◽  
Author(s):  
Jihoe Kim ◽  
Daniel Darley ◽  
Thorsten Selmer ◽  
Wolfgang Buckel
Keyword(s):  
Amino Acid ◽  
Clostridium Difficile ◽  
Coenzyme A ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Transferase Activity ◽  
Strictly Anaerobic ◽  
Radical Intermediates ◽  
Coa Transferase ◽  
Genes Encoding

ABSTRACT The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO2 and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to d-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (Km = 68 μM, k cat = 31 s−1) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate.

scholarly journals Mechanism of Resistance to Several Antimicrobial Agents in Salmonella Clinical Isolates Causing Traveler's Diarrhea

2004 ◽  
Vol 48 (10) ◽  
pp. 3934-3939 ◽  
Author(s):  
Roberto Cabrera ◽  
Joaquím Ruiz ◽  
Francesc Marco ◽  
Inés Oliveira ◽  
Margarita Arroyo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Antimicrobial Resistance ◽  
Nalidixic Acid ◽  
Molecular Mechanisms ◽  
Antimicrobial Agents ◽  
Mechanisms Of Resistance ◽  
Traveler’S Diarrhea ◽  
Genes Encoding ◽  
Dihydrofolate Reductases ◽  
Traveler's Diarrhea

ABSTRACT The evolution of antimicrobial resistance in Salmonella isolates causing traveler's diarrhea (TD) and their mechanisms of resistance to several antimicrobial agents were analyzed. From 1995 to 2002, a total of 62 Salmonella strains were isolated from stools of patients with TD. The antimicrobial susceptibility to 12 antibiotics was determined, and the molecular mechanisms of resistance to several of them were detected as well. The highest levels of resistance were found against tetracycline and ampicillin (21 and 19%, respectively), followed by resistance to nalidixic acid (16%), which was mainly detected from 2000 onward. Molecular mechanisms of resistance were analyzed in 16 isolates. In these isolates, which were resistant to ampicillin, two genes encoding β-lactamases were detected: oxa-1 (one isolate) and tem-like (seven isolates [in one strain concomitantly with a carb-2]). Resistance to tetracycline was mainly related to tetA (five cases) and to tetB and tetG (one case each). Resistance to chloramphenicol was related to the presence of the floR and cmlA genes and to chloramphenicol acetyltransferase activity in one case each. Different genes encoding dihydrofolate-reductases (dfrA1, dfrA12, dfrA14, and dfrA17) were detected in trimethoprim-resistant isolates. Resistance to nalidixic acid was related to the presence of mutations in the amino acid codons 83 or 87 of the gyrA gene. Further surveillance of the Salmonella spp. causing TD is needed to detect trends in their resistance to antimicrobial agents, as we have shown in our study with nalidixic acid. Moreover, such studies will lead to better treatment and strategies to prevent and limit their spread.

scholarly journals Identical Penicillin-Binding Domains in Penicillin-Binding Proteins of Streptococcus pneumoniae Clinical Isolates with Different Levels of β-Lactam Resistance

2005 ◽  
Vol 49 (7) ◽  
pp. 2895-2902 ◽  
Author(s):  
Laurent Chesnel ◽  
Raphaël Carapito ◽  
Jacques Croizé ◽  
Otto Dideberg ◽  
Thierry Vernet ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Proteins ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Penicillin G ◽  
Resistance Level ◽  
Penicillin Binding Proteins ◽  
Binding Domains ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT We have sequenced the penicillin-binding domains of the complete repertoire of penicillin-binding proteins and MurM from 22 clinical isolates of Streptococcus pneumoniae that span a wide range of β-lactam resistance levels. Evidence of mosaicism was found in the genes encoding PBP 1a, PBP 2b, PBP 2x, MurM, and, possibly, PBP 2a. Five isolates were found to have identical PBP and MurM sequences, even though the MICs for penicillin G ranged from 0.25 to 2.0 mg/liter. When the sequences encoding PBP 1a, PBP 2b, and PBP 2x from one of these isolates were used to transform laboratory strain R6, the resulting strain had a resistance level higher than that of the less resistant isolates carrying that PBP set but lower than that of the most resistant isolates carrying that PBP set. This result demonstrates that if the R6 strain is arbitrarily defined as the standard genotype, some wild genetic backgrounds can either increase or decrease the PBP-based resistance phenotype.

scholarly journals Identification of novel pneumolysin alleles from paediatric carriage isolates of Streptococcus pneumoniae

2010 ◽  
Vol 59 (7) ◽  
pp. 808-814 ◽  
Author(s):  
J. M. C. Jefferies ◽  
A. S. Tocheva ◽  
H. Rubery ◽  
J. Bennett ◽  
J. Garland ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Virulence Factor ◽  
Multilocus Sequence Typing ◽  
Clinical Isolates ◽  
Vaccine Candidates ◽  
Major Virulence Factor ◽  
Novel Alleles ◽  
Potential Use

Pneumolysin (Ply) is a major virulence factor of Streptococcus pneumoniae and is produced by all known clinical isolates of pneumococci. Pneumolysin toxoids are being considered as vaccine candidates. We investigated the diversity of pneumolysin among 194 nasopharyngeal pneumococci characterized by serotyping and multilocus sequence typing (MLST). Eight Ply protein alleles were identified, four of which were novel. The 4 novel alleles varied at 10 different amino acid positions, from a total of 147, 3 of these substitutions have been previously reported in different combinations. The protein allele correlated closely with MLST. It is critical that the presence of pneumolysin variants is considered with regards to the potential use of Ply in future vaccine formulations, as variation in Ply amino acid sequence may influence the immunogenicity of vaccines based on the presence of an individual Ply allele.

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