Point Mutations in the folP Gene Partly Explain Sulfonamide Resistance of Streptococcus mutans.

Cotrimoxazole inhibits dhfr and dhps and reportedly selects for drug resistance in pathogens. Here, Streptococcus mutans isolates were obtained from saliva of HIV/AIDS patients taking cotrimoxazole prophylaxis in Uganda. The isolates were tested for resistance to cotrimoxazole and their folP DNA (which encodes sulfonamide-targeted enzyme dhps) cloned in pUC19. A set of recombinant plasmids carrying different point mutations in cloned folP were separately transformed into folP-deficient Escherichia coli. Using sulfonamide-containing media, we assessed the growth of folP-deficient bacteria harbouring plasmids with differing folP point mutations. Interestingly, cloned folP with three mutations (A37V, N172D, R193Q) derived from Streptococcus mutans 8 conferred substantial resistance against sulfonamide to folP-deficient bacteria. Indeed, change of any of the three residues (A37V, N172D, and R193Q) in plasmid-encoded folP diminished the bacterial resistance to sulfonamide while removal of all three mutations abolished the resistance. In contrast, plasmids carrying four other mutations (A46V, E80K, Q122H, and S146G) in folP did not similarly confer any sulfonamide resistance to folP-knockout bacteria. Nevertheless, sulfonamide resistance (MIC = 50 µ M) of folP-knockout bacteria transformed with plasmid-encoded folP was much less than the resistance (MIC = 4 mM) expressed by chromosomally-encoded folP. Therefore, folP point mutations only partially explain bacterial resistance to sulfonamide.

Antimalarial resistance and DHFR/DHPS genotypes of Plasmodium falciparum three years after introduction of sulfadoxine-pyrimethamine and amodiaquine in rural Tanzania.

We assessed the efficacy of sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) and DHFR/DHPS genotypes of Plasmodium falciparum in rural Tanzania, 3 years after their introduction as first- and second-line treatments for uncomplicated malaria, respectively. Under five children with uncomplicated malaria were given standard treatments of either SP (n=66) or AQ (n=30) and treatment outcomes after 14 and 28 days were determined. Total treatment failure of 18 and 42.5% was observed for SP on days 14 and 28, respectively. For AQ, total treatment failure of 27 and 53% was found on day 14 and 28, respectively. On day 14, significantly lower SP total treatment failures were observed in 2004 compared with results from a study conducted in 1999 in the same location. No relationship was detected between clinical outcome and DHFR/DHPS genotypes, but the point mutation prevalence in parasites was higher than in 1999. Pre-treatment blood levels of SP were detected in a quarter of the study children: less than expected. We report unacceptably high levels of total treatment failures, both for first- and second-line treatments for uncomplicated malaria in Tanzania 3 years after their introduction, supporting the decision to replace them with artemisinin-based combination therapy.

Patterns of resistance and DHFR/DHPS genotypes of Plasmodium falciparum in rural Tanzania prior to the adoption of sulfadoxine-pyrimethamine as first-line treatment.

A study was carried out to assess the patterns of resistance and occurrence of DHFR/DHPS genotypes of Plasmodium falciparum prior to the adoption of sulfadoxine-pyrimethamine (SP) as first-line treatment for uncomplicated malaria in Tanzania. Children under five years (n = 117) with clinical, uncomplicated malaria were randomly allocated to standard treatments of either chloroquine (CQ) (25 mg/kg) or SP (25 mg sulfadoxine and 1.25 mg pyrimethamine/kg). Patients were monitored for 28 days. Clinical recovery was achieved in 98% (n = 58) and 90% (n = 59) of the patients in the SP and CQ groups, respectively. Parasitologically, 14% of the patients in the SP group and 51% in the CQ group exhibited RII/RIII resistance. When relating pre-treatment blood drug levels to treatment outcome and the degree of parasite resistance to the number of mutations, no relationships could be detected. There was an overall significant increase in haemoglobin levels from day 0 to day 28 in both patient groups. Sulfadoxine-pyrimethamine produced an acceptable clinical response but the high degree of parasitological resistance (RII/RIII) observed two years prior to the introduction of the drug as first-line treatment is of concern, especially considering the long half-lives of sulfadoxine and pyrimethamine.

Amino acid repetitions in the dihydropteroate synthase of Streptococcus pneumoniae lead to sulfonamide resistance with limited effects on substrate K(m).

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. pneumoniae isolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile(66) and Glu(67) 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 K(i) for sulfathiazole but changed the K(m) for p-aminobenzoic acid only 2.5-fold and did not significantly change the K(m) 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 Ser(61) 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.

Computational predictions of binding affinities to dihydrofolate reductase: synthesis and biological evaluation of methotrexate analogues.

The relative binding affinities to human dihydrofolate reductase of four new potential antifolates, containing ester linkages between the two aromatic systems, were estimated by free energy perturbation simulations. The ester analogue, predicted to exhibit the highest binding affinity to human dihydrofolate reductase, and a reference ester (more structurally related to methotrexate) were synthesized. As deduced from the measured IC(50) values, the calculated ranking of the ligands was correct although a greater difference in affinity was indicated by the experimental measurements. Among the new antifolates the most potent inhibitor exhibited a similar pharmacokinetic profile to methotrexate but lacked activity in a complex antiarthritic model in rat in vivo.

Molecular epidemiology of Plasmodium falciparum antifolate resistance in Vietnam: genotyping for resistance variants of dihydropteroate synthase and dihydrofolate reductase.

Using PCR techniques, we analysed the dihydropteroate synthase (DHPS) mutations associated with sulphonamide resistance and the dihydrofolate reductase (DHFR) mutations associated with resistance to pyrimethamine and cycloguanil in samples from Plasmodium falciparum-infected Vietnamese patients. Of the 40 samples analysed, 39 had DHFR mutations associated with high level resistance to pyrimethamine, whereas only three had mutations at position 164, which is linked to cross resistance to both DHFR inhibitors. The DHPS, 437Gly variant associated with very mild resistance to sulphadoxine was found in 38 out of the 40 samples. Of seven samples resistant to Fansidar in vivo, only two were fully explained by the currently documented DHPS mutations. The treatment failure could be due to a high level of pyrimethamine resistance caused by the detected mutations. Most patients, however, were cured with a single dose of Fansidar in spite of the high number of resistance mutations found.

Sulfonamide resistance in Streptococcus pyogenes is associated with differences in the amino acid sequence of its chromosomal dihydropteroate synthase.

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.

Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins.

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine-sulfadoxine; PYR-SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.

Adaptation to sulfonamide resistance in Neisseria meningitidis may have required compensatory changes to retain enzyme function: kinetic analysis of dihydropteroate synthases from N. meningitidis expressed in a knockout mutant of Escherichia coli.

Previously, the effects of three point mutations (at amino acid positions 31, 84, and 194) in the gene coding for a sulfonamide-resistant dihydropteroate synthase of Neisseria meningitidis were analyzed by site-directed mutagenesis. Changes at positions 31 and 194 abolished the phenotypic expression of sulfonamide resistance, while a change at position 84 appeared to be neutral. These studies are here extended to correlate the alterations in phenotype with effects on enzyme kinetics by expressing the cloned meningococcal genes in an Escherichia coli strain that had its dhps gene partially deleted and replaced by a resistance determinant. The most dramatic effects were produced by mutations at position 31. A change from the Leu found in the resistant isolate to a Phe (the residue found in sensitive isolates) led to a 10-fold decrease in the Km and a concomitant drop in the Ki. Changes at position 194 also affected both the Km and Ki but not to the same extent as mutations at position 31. Changing position 84 altered the Km only slightly but significantly. This latter change was interpreted as a compensatory change modulating the function of the enzyme. In another type of resistance gene, 2 amino acid residues, proposed to be an insertion, were deleted, resulting in a sensitive enzyme. However, the resulting Km was raised 10-fold, suggesting that compensatory changes have accumulated in this type of resistance determinant as well. This resistance gene differs by as much as 10% from the sensitive isolates, which makes identification of important mutations difficult.

Plasmodium falciparum: mutation pattern in the dihydrofolate reductase-thymidylate synthase genes of Vietnamese isolates, a novel mutation, and coexistence of two clones in a Thai patient.

Pyrimethamine and cycloguanil resistance of Plasmodium falciparum has been linked to mutations in the dihydrofolate reductase (dhfr) portion of the dhfr-ts gene. In this paper, the DNA sequence of the dhfr-ts gene of 50 isolates from Vietnam and 2 clones (T9/94 and T9/96) isolated from a malaria patient from Thailand have been analyzed. A comparison between these isolates and clones showed differential mutation patterns. Forty-eight isolates were found to consist of mutations associated with Pyr. A novel leucine mutation at position 140 was found in the isolate VP8 and in clone T9/94. The isolate VP8 and the clone T9/94 were found to also have the characteristic changes at positions 16 (Val) and 108 (Thr) that have been found in cycloguanil-resistant isolates. The isolate VP35 was shown to be resistant to both antifolates, while the clone T9/96 was found to be sensitive to both antifolates and to have a sequence identical to that of wild-type dhfr-ts. The two clones from a single patient showed the coexistence of resistant and sensitive clones in the absence of treatment by antifolates. Since cycloguanil resistance seems to be rare in Vietnam, cycloguanil alone or in combination with other antimalarial agents might be an alternative for treatment and prophylaxis, even in areas with high resistance to pyrimethamine.

Rapid detection of pyrimethamine susceptibility of Plasmodium falciparum by restriction endonuclease digestion of the dihydrofolate reductase gene.

A rapid and simple method to detect pyrimethamine susceptibility of Plasmodium falciparum by analyzing DNA from whole blood is presented. Samples from cultured isolates and from patients infected with P. falciparum were spotted onto filter paper disks, dried, and stored for subsequent analyses. After extracting the P. falciparum DNA using Chelex-100 ion-chelating resin (Bio-Rad, Richmond, CA), the polymerase chain reaction (PCR) was used to amplify the dihydrofolate reductase (dhfr) gene. The PCR product of 727 basepairs was digested with the Alu I restriction endonuclease to detect whether the isolates were sensitive or resistant to the antimalarial drugs pyrimethamine and cycloguanil. This reaction endonuclease digests only DNA from pyrimethamine-sensitive parasites because the recognition locus of Alu I is changed by mutations giving rise to pyrimethamine and cycloguanil resistance. This method is simple and sensitive and could therefore bu used to study the epidemiology of pyrimethamine resistant in P. falciparum. The DHFR gene of pyrimethamine-resistance clones from Vietnamese patients showed three amino acid changes that were previously found in pyrimethamine-resistant isolates. Two other clones, T9/94 and T9/96, originally isolated from a single malaria patient from Thailand, had different DHFR gene sequences. The nucleotide sequence of the DHFR gene from T9/96 was identical to the wild-type DHFR sequence, whereas T9/94 possessed amino acid substitutions at positions 16 and 108 that have been described in cycloguanil-resistant parasites.

Sulfonamide resistance in Neisseria meningitidis as defined by site-directed mutagenesis could have its origin in other species.

Sulfonamide resistance in Neisseria meningitidis is mediated by altered forms of the chromosomal gene for the drug target enzyme dihydropteroate synthase. Sulfonamides have been used for decades both for prophylaxis and the treatment of meningococcal disease, and resistance is common. Two types of resistance determinants have been identified, and regions important for drug insusceptibility to the corresponding enzyme have been defined by site-directed mutagenesis. Both types of resistance traits have spread among strains of N. meningitidis of different serogroups and serotypes, and the large differences at the nucleotide level in a comparison of the resistance genes with the dhps genes of susceptible meningococci indicate the origin of one or maybe both types in other Neisseria species. One sulfonamide-sensitive strain of N. meningitidis was found to have a mosaic dhps gene with a central part identical to the corresponding part of a gonococcal strain. This observation supports the idea of an interspecies transfer of genetic material in Neisseria species as a mechanism for the development of chromosomally mediated resistance.

PCR amplicon restriction endonuclease analysis of the chromosomal dhps gene of Neisseria meningitidis: a method for studying spread of the disease-causing strain in contacts of patients with meningococcal disease.

We tested two sets of primers derived from the dhps gene of Neisseria meningitidis for the amplification of meningococcal DNA by PCR. Both the NM1-NM6 primers and the NM3-NM6 primers amplified dhps DNA from all of the meningococci included in the study, resulting, in most cases, in amplicons of 0.70 and 0.23 kb, respectively. Also, dhps DNAs of N. gonorrhoeae and some commensals were amplified but Haemophilus influenzae, Streptococcus pneumoniae, and Escherichia coli DNAs were not. By PCR amplicon restriction endonuclease analysis (AREA) of the larger amplicon, we could differentiate between individual strains of N. meningitidis. Following two cases of meningococcal disease, we used PCR AREA to identify healthy contacts carrying the disease-causing strain. We conclude that PCR AREA is a useful method for meningococcal strain differentiation and that it has potential as a method for studying the spread of a disease-causing strain in an affected population. The method is quicker and easier to perform and interpret than chromosomal DNA fingerprinting.

Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements.

Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086. Several characteristics suggest that this element is a transposon, which we call Tn5090. Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins. The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7. Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences. Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein. The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end. Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly. Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism. The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements.

The 3' conserved segment of integrons contains a gene associated with multidrug resistance to antiseptics and disinfectants.

Nucleotide sequence analysis of ORF1 from the integron on the broad-host-range plasmid R751 revealed that the first 94 of 110 codons of ORF1 from R751 are identical to ORF4, an open reading frame from the 3' conserved segment of other integrons found in gram-negative bacteria, after which point they diverged completely. The predicted products of both ORF1 and ORF4 share homology with the multidrug exporter QacC. Phenotypic analysis revealed that ORF1 specifies a resistance profile to antiseptics and disinfectants almost identical to that of qacC, whereas ORF4 specifies much lower levels of resistance to these compounds. ORF4, whose product lacks the C-terminal 16 amino acids of the ORF1 protein, may have evolved by the interruption of ORF1 from the insertion of a DNA segment carrying a sulI sulfonamide resistance determinant. Hence, ORF1 was designated qacE, and its partially functional deletion derivative, ORF4, was designated qacE delta 1. Fluorimetric experiments indicated that the mechanism of resistance mediated by QacE, the protein specified by qacE, is active export energized by proton motive force. Amino acid sequence comparisons revealed that QacE is related to a family of small multidrug export proteins with four transmembrane segments.

Characterization of transposon Tn5086, carrying the site-specifically inserted gene dhfrVII mediating trimethoprim resistance.

Two different enteric plasmids of widely separate origins were observed to carry a new 15.3-kb trimethoprim resistance transposon, Tn5086, also mediating resistance to mercuric ions and to a low level of sulfonamide. The trimethoprim resistance gene characterized from Tn5086 was found to be distinct from those found earlier and was designated type VII. Molecular analysis demonstrated that Tn5086 is closely related to Tn21. The internal part of Tn21 and Tn5086, the element referred to as the integron, was found to be different. First, the integron of Tn5086 contains a 0.62-kb cassette formed by the trimethoprim resistance gene dhfrVII and its immediate surroundings instead of the 0.86-kb aadA1 cassette of Tn21. Second, the integron of Tn5086 lacks a 4.2-kb segment 3' of sulI in Tn21. The dhfrVII gene commences with a UUG codon but was otherwise seen to be markedly related to the cassette genes dhfrI, dhfrV, and dhfrVI. The four related dihydrofolate reductases of 157 amino acids encoded by these genes contain a glutamate instead of the aspartic acid residue found at position 27 of the active center of the chromosomal enzyme from Escherichia coli.

Transformational exchanges in the dihydropteroate synthase gene of Neisseria meningitidis: a novel mechanism for acquisition of sulfonamide resistance.

The nucleotide sequences of the chromosomal dihydropteroate synthase (dhps) genes in sulfonamide-susceptible and sulfonamide-resistant strains of Neisseria meningitidis of serogroups A, B and C were determined. The molecular weights and the amino acid sequences showed similarity to those of all other known dihydropteroate synthase polypeptides. Sequence comparison of the N. meningitidis dhps genes indicated horizontal transfer of DNA segments rather than point mutations as the cause for resistance in meningococci. The dhps genes in three of four sulfonamide-resistant meningococci contained identical central regions of 424 bp. Compared with the corresponding genes in susceptible strains, each central region included an insert of 6 bp. In one of the sulfonamide-resistant strains, the dhps gene was similar to the corresponding genes in the sensitive strains in its NH2-terminal and C-terminal parts. Its central region, however, was identical to the corresponding regions of two of the other resistant genes, and thus it could be seen as a hybrid dhps gene. Transformation experiments and mapping of transformed dhps genes indicated the existence of a novel mechanism for the dissemination of sulfonamide resistance in N. meningitidis. The origin of the resistance-mediating segment of the gene is unknown, but hybridization results showed the presence of homologous dhps genes in Neisseria gonorrhoeae and N. lactamica but not in N. subflava or Branhamella catarrhalis.

Genetic analyses of sulfonamide resistance and its dissemination in gram-negative bacteria illustrate new aspects of R plasmid evolution.

In contrast to what has been observed for many other antibiotic resistance mechanisms, there are only two known genes encoding plasmid-borne sulfonamide resistance. Both genes, sulI and sulII, encode a drug-resistant dihydropteroate synthase enzyme. In members of the family Enterobacteriaceae isolated from several worldwide sources, plasmid-mediated resistance to sulfonamides could be identified by colony hybridization as being encoded by sulI, sulII, or both. The sulI gene was in all cases found to be located in the newly defined, mobile genetic element, recently named an integron, which has been shown to contain a site-specific recombination system for the integration of various antibiotic resistance genes. The sulII gene was almost exclusively found as part of a variable resistance region on small, nonconjugative plasmids. Colony hybridization to an intragenic probe, restriction enzyme digestion, and nucleotide sequence analysis of small plasmids indicated that the sulII gene and contiguous sequences represent an independently occurring region disseminated in the bacterial population. The sulII resistance region was bordered by direct repeats, which in some plasmids were totally or partially deleted. The prevalence of sulI and sulII could thus be accounted for by their stable integration in transposons and in plasmids that are widely disseminated among gram-negative bacteria.