Göte Swedberg

Trimethoprim and sulfonamide resistance.

Trimethoprim (TMP) and sulfonamides (SULs) are synthetic antibacterial agents. The first SUL compounds were used in 1932, whereas TMP is a relatively new compound first used in 1962 in England (69). Since 1968, TMP has been used in combination with SULs because the combination of TMPSULs was supposed to be synergistic in vitro (18). However, clinical experience suggests that TMP-SUL combinations have no clear synergism in vivo (2, 16, 112). In the 1970s, TMP alone came into use, first for the prophylaxis of urinary tract infections and later for the treatment of acute urinary tract infections as well (81). TMP and SULs share both a wide antibacterial spectrum including common urinary tract pathogens (Escherichia coli and other members of the family Enterobacteriaceae), respiratory tract pathogens (Streptococcus pneumoniae, Haemophilus influenzae, and in combination, Moraxella catarrhalis), skin pathogens (Staphylococcus aureus), as well as certain enteric pathogens (E. coli and Shigella spp.). Because of the wide range of clinical indications, TMP-SUL combinations have been used extensively everywhere in the world. In addition, both compounds are relatively inexpensive, a fact that allows for the use of these drugs outside of developed countries. Today, the most important fear is the development of bacterial resistance to TMP and SULs. To counter bacterial resistance it is essential to understand the molecular background of resistance mechanisms. Analysis of TMP and SUL resistance determinants in clinical bacteria has already revealed new recombination mechanisms that have an impact on spread of the resistance in general. As synthetic antimicrobial agents, TMP and SULs are also examples of agents that bacteria have not met previously, and to which they can develop resistance; this excludes resistance mechanisms related to antibiotic-producing organisms. In this minireview, we describe the current knowledge of TMP and SUL resistance in major bacterial pathogens and review the TMP and SUL resistance mechanisms.

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.

Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21

SummaryA new gene for trimethoprim resistance, dhfrV, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type I. The sequenced surroundings of dhfrV in plasmid pLMO20, were found to be almost identical with genetic areas surrounding resistance genes in transposon Tn21 and in R plasmid R388. The trimethoprim resistance genes of pLMO20 and R388 and the spectinomycin resistance gene of Tn21 could be regarded as having been inserted, by recombination, into an evolutionary older structure containg the sulfonamide resistance gene, sulI. The latter gene was sequenced and found to correspond to a peptide of 279 amino acids and with a molecular weight of 30126 daltons. The inserted genes were found to be governed by a promoter situated in the highly conserved structure and also controlling expression of sulI. The insertion points of the different resistance genes were precisely defined, and at the 3′ ends of the inserted genes inverted repeats allowing the formation of stem and loop structures were found. Similar structures were found at the 3′ ends of the antibiotic resistance genes in Tn7, which could indicate similar recombination mechanisms to be effective in the evolutionary construction of all these different resistance elements.

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. Images

RSF1010 and a conjugative plasmid contain sulII, one of two known genes for plasmid-borne sulfonamide resistance dihydropteroate synthase.

The nucleotide sequence of the type II sulfonamide resistance dihydropteroate synthase (sulII) gene was determined. The molecular weight determined by maxicells was 30,000, and the predicted molecular weight for the polypeptide was 28,469. Comparison with the sulI gene encoded by Tn21 showed 57% DNA similarity. The sulII-encoded polypeptide has 138 of 271 amino acids in common with the polypeptide encoded by sulI. The sulII gene is located on various IncQ (broad-host-range) plasmids and other small nonconjugative resistance plasmids. Detailed restriction maps were constructed to compare the different plasmids in which sulII is found. The large conjugative plasmid pGS05 and the IncQ plasmid RSF1010 contained identical nucleotide sequences for the sulII gene. This type of sulfonamide resistance is very frequently found among gram-negative bacteria because of its efficient spread to various plasmids. Images

Evidence of Plasmodium falciparum malaria resistant to atovaquone and proguanil hydrochloride: case reports

The increased spread of drug resistant malaria highlights the need for alternatives for treatment and chemoprophylaxis. The combination of atovaquone and proguanil hydrochloride (Malarone, GlaxoSmithKline, NC) has shown high efficacy against Plasmodium falciparum with only mild side effects and has been registered for use in several countries, including Denmark, Germany, Sweden, the United Kingdom, and the United States.1 Treatment failures have been attributed to suboptimal dosage, reinfections, or to a point mutation in the cytochrome b gene. 1 2 Bioavailability of atovaquone depends on the concomitant intake of a fatty diet, yet drug concentrations were not analysed in these reports. We provide evidence of resistance in two patients treated with atovaquone and proguanil hydrochloride for P falciparum infection. View this table: Details of three patients treated with atovaquone and proguanil hydrochloride (Malarone; GlaxoSmithKline) for Plasmodium …

Impaired fitness of drug-resistant malaria parasites: evidence and implication on drug-deployment policies

Malaria, a leading parasitic disease, inflicts an enormous toll on human lives and is caused by protozoal parasites belonging to the genus Plasmodium. Antimalarial drugs targeting essential biochemical processes in the parasite are the primary resources for management and control. However, the parasite has established mutations, substantially reducing the efficacy of these drugs. First-line therapy is faced the with the consistent evolution of drug-resistant genotypes carrying these mutations. However, drug-resistant genotypes are likely to be less fit than the wild-type, suggesting that they might disappear by reducing the volume of drug pressure. A substantial body of epidemiological evidence confirmed that the frequency of resistant genotypes wanes when active drug selection declines. Drug selection on the parasite genome that removes genetic variation in the vicinity of drug-resistant genes (hitch-hiking) is common among resistant parasites in the field. This can further disadvantage drug-resistant strains and limit their variability in the face of a mounting immune response. Attempts to provide unequivocal evidence for the fitness cost of drug resistance have monitored the outcomes of laboratory competition experiments of deliberate mixtures of sensitive and resistant strains, in the absence of drug pressure, using isogenic clones produced either by drug selection or gene manipulation. Some of these experiments provided inconclusive results, but they all suggested reduced fitness of drug-resistant clones in the absence of drug pressure. In addition, biochemical analyses provided clearer information demonstrating that the mutation of some antimalarial-targeted enzymes lowers their activity compared with the wild-type enzyme. Here, we review current evidences for the disadvantage of drug-resistance mutations, and discuss some strategies of drug deployment to maximize the cost of resistance and limit its spread.

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

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.

Point mutations in the dihydropteroate synthase gene causing sulfonamide resistance.

Sulfonamides act by competing with p-aminobenzoic acid (PABA) in the formation of dihydropteroate by the enzyme dihydropteroate synthase1. In many bacteria, as well as protozooans and fungi, this inhibition leads to a block in the synthesis of dihydrofolate, and eventually to a stop in DNA synthesis due to lack of dTTP.

Efficacy of artemether-lumefantrine in treatment of malaria among under-fives and prevalence of drug resistance markers in Igombe-Mwanza, north-western Tanzania.

BackgroundDrug resistance to anti-malarials is a major public health problem worldwide. This study aimed at establishing the efficacy of artemether-lumefantrine (ACT) in Igombe-Mwanza, north-western Tanzania after a few years of ACT use, and establish the prevalence of mutations in key targets for artemisinin, chloroquine and sulphadoxine/pyrimetamine (SP) drugs.MethodsA prospective single cohort study was conducted at Igombe health centre using artemether-lumefantrine combination therapy between February 2010 and March 2011. The follow-up period was 28 days and outcome measures were according to WHO guidelines. Blood was collected on Whatman filter paper for DNA analysis. DNA extraction was done using TRIS-EDTA method, and mutations in Pfcrt, Pfmdr 1, Pfdhfr, Pfdhps and Pfatp 6 were detected using PCR-RFLP methods established previously.ResultsA total of 103 patients completed the 28 days follow-up. The mean haemoglobin was 8.9 g/dl (range 5.0 to 14.5 g/dl) and mean parasite density was 5,608 parasites/μl. Average parasite clearance time was 34.7 hours and all patients cleared the parasites by day 3. There was no early treatment failure in this study. Late clinical failure was seen in three (2.9%) patients and late parasitological failure (LPF) was seen in two (1.9%). PCR-corrected LPF was 1% and adequate clinical and parasitological response was 96%. The majority of parasites have wild type alleles on pfcrt 76 and pfmdr 1 86 positions being 87.8% and 93.7% respectively. Mutant parasites predominated at pfdhfr gene at the main three positions 108, 51 and 59 with prevalence of 94.8%, 75.3% and 82.5% respectively. Post-treatment parasites had more wild types of pfdhps at position 437 and 540 than pre-treatment parasites. No mutation was seen in pfatp6 769 in re-infecting or recrudescing parasites.ConclusionThe efficacy of artemether-lumefantrine for treatment of uncomplicated malaria is still high in the study area although the rate of re-infection is higher than previously reported. Parasite clearance after 48 hours was lower compared to previous studies. The prevalence of wild type allele pfcrt 76 K and pfmdr 1 86 N was high in the study area while markers for SP resistance is still high. Artemether-lumefantrine may be selecting for wild type alleles on both positions (437 and 540) of pfdhps.