Mechanism of sulfonamide resistance in clinical isolates of Streptococcus pneumoniae.

1997 ◽  
Vol 41 (10) ◽  
pp. 2121-2126 ◽  
Author(s):  
J P Maskell ◽  
A M Sefton ◽  
L M Hall
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genetic Basis ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Gene Exchange ◽  
Gene Encoding ◽  
Dihydropteroate Synthase ◽  
Loss Of Resistance ◽  
Sulfonamide Resistance

The genetic basis of sulfonamide resistance in six clinical isolates of Streptococcus pneumoniae was demonstrated to be 3- or 6-bp duplications within sulA, the chromosomal gene encoding dihydropteroate synthase. The duplications all result in repetition of one or two amino acids in the region from Arg58 to Tyr63, close to but distinct from the sul-d mutation, a duplication previously reported in a resistant laboratory strain (P. Lopez, M. Espinosa, B. Greenberg, and S. A. Lacks, J. Bacteriol. 169:4320-4326, 1987). Six sulfonamide-susceptible clinical isolates lacked such duplications. The role of the duplications in conferring sulfonamide resistance was confirmed by transforming 319- or 322-bp PCR fragments into the chromosome of a susceptible recipient. Two members of a clone of serotype 9V, one susceptible and one resistant to sulfonamide, which are highly related by other criteria, were shown to have sulA sequences that differ in 7.2% of nucleotides in addition to the duplication responsible for resistance. It is postulated that horizontal gene exchange has been involved in the acquisition (or loss) of resistance within this clone. However, five of the six resistant isolates have distinct duplications and other sequence polymorphisms, suggesting that resistance has arisen independently on many occasions.

scholarly journals Novel Expansions of the Gene Encoding Dihydropteroate Synthase in Trimethoprim-Sulfamethoxazole-ResistantStreptococcus pneumoniae

1999 ◽  
Vol 43 (9) ◽  
pp. 2225-2230 ◽  
Author(s):  
Thanugarani Padayachee ◽  
Keith P. Klugman
Keyword(s):  
South African ◽  
Specific Region ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Wild Type ◽  
Gene Encoding ◽  
Dihydropteroate Synthase ◽  
Sulfonamide Resistance ◽  
Resistant Strains ◽  
Chromosomal Mutations

ABSTRACT A study of eight sulfonamide-resistant clinical isolates ofStreptococcus pneumoniae revealed chromosomal mutations within the gene encoding dihydropteroate synthase that play a role in conferring resistance to sulfamethoxazole. The presence of thesuld mutation, found previously only in a laboratory mutant, was shown to occur in three of the wild-type clinical isolates. The duplication of Ser61, the other previously defined mutation in the dihydropteroate synthase gene of S. pneumoniae, was observed in only one of the isolates characterized. We report two previously unidentified amino acid alterations, namely, a duplication of Arg58 and Pro59 and an insertion of an arginine residue between Gly60 and Ser61 in trimethoprim-sulfamethoxazole-resistant strains. The significance of these mutations was confirmed by site-directed mutagenesis and by the transformation of a susceptible strain of S. pneumoniae to sulfamethoxazole resistance. Two resistant isolates did not contain any mutations within the gene encoding dihydropteroate synthase. The results presented suggest the independent generation of resistant mutations among South African clinical isolates. It is also proposed that the mechanism of sulfonamide resistance in S. pneumoniae involves the expansion of a specific region within dihydropteroate synthase, which probably forms part of the sulfonamide binding site.

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 Sulfonamide Resistance in Streptococcus pyogenes Is Associated with Differences in the Amino Acid Sequence of Its Chromosomal Dihydropteroate Synthase

1998 ◽  
Vol 42 (5) ◽  
pp. 1062-1067 ◽  
Author(s):  
Göte Swedberg ◽  
Signe Ringertz ◽  
Ola Sköld
Keyword(s):  
Amino Acid ◽  
Streptococcus Pyogenes ◽  
Gtp Cyclohydrolase ◽  
Gene Encoding ◽  
Additional Indication ◽  
Kinetic Measurements ◽  
Dihydropteroate Synthase ◽  
Development Of Resistance ◽  
Sulfonamide Resistance ◽  
Genes Encoding

ABSTRACT Sulfonamide resistance in recent isolates of Streptococcus pyogenes was found to be associated with alterations of the chromosomally encoded dihydropteroate synthase (DHPS). There were 111 different nucleotides (13.8%) in the genes found in susceptible and resistant isolates, respectively, resulting in 30 amino acid changes (11.3%). These substantial changes suggested the possibility of a foreign origin of the resistance gene, in parallel to what has already been found for sulfonamide resistance in Neisseria meningitidis. The gene encoding DHPS was linked to at least three other genes encoding enzymes of the folate pathway. These genes were in the order GTP cyclohydrolase, dihydropteroate synthase, dihydroneopterin aldolase, and hydroxymethyldihydropterin pyrophosphokinase. The nucleotide differences in genes from resistant and susceptible strains extended from the beginning of the GTP cyclohydrolase gene to the end of the gene encoding DHPS, an additional indication for gene transfer in the development of resistance. Kinetic measurements established different affinities for sulfathiazole for DHPS enzymes isolated from resistant and susceptible strains.

scholarly journals Mechanisms of Macrolide Resistance in Clinical Pneumococcal Isolates in France

2001 ◽  
Vol 45 (2) ◽  
pp. 636-638 ◽  
Author(s):  
Frederic Fitoussi ◽  
Catherine Doit ◽  
Pierre Geslin ◽  
Naima Brahimi ◽  
Edouard Bingen
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genetic Basis ◽  
Macrolide Resistance ◽  
Clinical Isolates ◽  
Resistance Phenotype

ABSTRACT The genetic basis of macrolide resistance was investigated in a collection of 48 genotypically unrelated clinical isolates ofStreptococcus pneumoniae obtained between 1987 and 1997 in France. All strains were resistant to erythromycin, clindamycin, and streptogramin B, exhibiting a macrolide-lincosamide-streptogramin B resistance phenotype, and harbored the erm(B) gene. None of the strains carried the mef(A) or erm(A) subclass erm(TR) gene.

scholarly journals Role of sarA in the Pathogenesis of Staphylococcus aureus Musculoskeletal Infection

2003 ◽  
Vol 71 (1) ◽  
pp. 516-523 ◽  
Author(s):  
Jon S. Blevins ◽  
Mohamed O. Elasri ◽  
Scott D. Allmendinger ◽  
Karen E. Beenken ◽  
Robert A. Skinner ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Septic Arthritis ◽  
Clinical Isolate ◽  
Murine Model ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Musculoskeletal Infection

ABSTRACT We recently demonstrated that mutation of sarA in clinical isolates of Staphylococcus aureus results in a phenotype that is distinct by comparison to sarA mutants generated in the laboratory strain RN6390 (J. S. Blevins, K. E. Beenken, M. O. Elasri, B. K. Hurlburt, and M. S. Smeltzer, Infect. Immun. 70:470-480, 2002). This raises the possibility that studies demonstrating that RN6390 sarA mutants are attenuated do not accurately reflect the role of sarA in the pathogenesis of staphylococcal disease. To test this hypothesis, we used a murine model of musculoskeletal infection to assess the virulence of sarA and agr mutants generated in a clinical isolate of S. aureus (UAMS-1). By using this model, we confirmed that mutation of sarA and/or agr results in a reduced capacity to cause both septic arthritis and osteomyelitis.

scholarly journals Mutation of traP in Staphylococcus aureus Has No Impact on Expression of agr or Biofilm Formation

2007 ◽  
Vol 75 (9) ◽  
pp. 4528-4533 ◽  
Author(s):  
Laura H. Tsang ◽  
Sonja T. Daily ◽  
Elizabeth C. Weiss ◽  
Mark S. Smeltzer
Keyword(s):  
Staphylococcus Aureus ◽  
Hemolytic Activity ◽  
Biofilm Formation ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Regulatory Role ◽  
Accessory Gene ◽  
Accessory Gene Regulator ◽  
Affect Expression

ABSTRACT To investigate the regulatory role of traP (target of RNAIII-activating peptide) in Staphylococcus aureus, we generated traP mutations in the clinical isolates UAMS-1 and USA300. In neither case did mutation of traP affect expression of the accessory gene regulator (agr) or the ability to form a biofilm. We were also unable to confirm that mutation of traP in the prototype 8325-4 laboratory strain RN6390 results in reduced expression of agr, reduced hemolytic activity, or an altered capacity to form a biofilm.

scholarly journals Expression of Efflux Pump Gene pmrA in Fluoroquinolone-Resistant and -Susceptible Clinical Isolates of Streptococcus pneumoniae

2002 ◽  
Vol 46 (3) ◽  
pp. 808-812 ◽  
Author(s):  
Laura J. V. Piddock ◽  
Maggie M. Johnson ◽  
S. Simjee ◽  
L. Pumbwe
Keyword(s):  
Streptococcus Pneumoniae ◽  
Reverse Transcriptase ◽  
Ethidium Bromide ◽  
Marked Effect ◽  
Efflux Pump ◽  
Northern Blotting ◽  
Clinical Isolates ◽  
Efflux Pump Inhibitor ◽  
Gene Encoding ◽  
Genes Expression

ABSTRACT Thirty-four ciprofloxacin-resistant (MIC ≥ 2 μg/ml) and 12 ciprofloxacin-susceptible clinical isolates of Streptococcus pneumoniae were divided into four groups based upon susceptibility to norfloxacin and the effect of reserpine (20 μg/ml). The quinolone-resistance-determining regions of parC, parE, gyrA, and gyrB of all ciprofloxacin-resistant clinical isolates were sequenced, and the activities of eight other fluoroquinolones, acriflavine, ethidium bromide, chloramphenicol, and tetracycline in the presence and absence of reserpine were determined. Despite a marked effect of reserpine upon the activity of norfloxacin, there were only a few isolates for which the activity of another fluoroquinolone was enhanced by reserpine. For most isolates the MICs of acriflavine and ethidium bromide were lowered in the presence of reserpine despite the lack of effect of this efflux pump inhibitor on fluoroquinolone activity. The strains that were most resistant to the fluoroquinolones were predominantly those with mutations in three genes. Expression of the gene encoding the efflux pump PmrA was examined by Northern blotting (quantified by quantitative competitive reverse transcriptase PCR) and compared with that of S. pneumoniae R6 and R6N. Within each group there were isolates that had high-, medium-, and low-level expression of this gene; however, increased expression was not exclusively associated with those isolates with a phenotype suggestive of an efflux mutant. These data suggest that there is another reserpine-sensitive efflux pump in S. pneumoniae that extrudes ethidium bromide and acriflavine but not fluoroquinolones.

Transformation of restriction endonuclease phenotype in Streptococcus pneumoniae

1982 ◽  
Vol 152 (1) ◽  
pp. 183-190
Author(s):  
C C Muckerman ◽  
S S Springhorn ◽  
B Greenberg ◽  
S A Lacks
Keyword(s):  
Streptococcus Pneumoniae ◽  
Restriction Endonuclease ◽  
Genetic Basis ◽  
Laboratory Strain ◽  
The Other ◽  
Unique Restriction ◽  
Different Strains ◽  
Spontaneous Conversion

The genetic basis of the unique restriction endonuclease DpnI, that cleaves only at a methylated sequence, 5'-GmeATC-3', and of the complementary endonuclease DpnII, which cleaves at the same sequence when it is not methylated, was investigated. Different strains of Streptococcus pneumoniae isolated from patients contained either DpnI (two isolates) or DpnII (six isolates). The latter strains also contained DNA methylated at the 5'-GATC-3' sequence. A restrictable bacteriophage, HB-3, was used to characterize the various strains and to select for transformants. One laboratory strain contained neither DpnI nor Dpn II. It was probably derived from a DpnI-containing strain, and its DNA was not methylated at 5'-GATC-3'. Cells of this strain were transformed to the DpnI restriction phenotype by DNA from a DpnI-containing strain and to the DpnII restriction phenotype by DNA from a DpnII-containing strain. Neither cross-transformation, that is, transformation to one phenotype by DNA from a strain of the other phenotype, nor spontaneous conversion was observed. Extracts of transformants to the new restriction phenotype were shown to contain the corresponding endonuclease.

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