Sulfonamides and trimethoprim

Figure 1. Phylogenetic tree showing the relationship between different dihydrofolate reductases, based on amino acid sequence alignment and parsimony analysis.

Resistance enzymes are marked by dfr and a number.

Modified from [27].

Sulfonamides and trimethoprim are illustrative examples of what has happened to cheap and efficient antibacterial agents under the evolution of resistance [1]. A description of the various ways in which bacteria have acquired and developed resistance against these drugs can also serve as a good and general illustration of the ingenious ways evolution has chosen to mediate and spread drug resistance, and may possibly also provide hints on how to counteract resistance development. Sulfonamides and trimethoprim are at the chronological extreme points in the development of antibacterial agents or antibiotics, which are the greatest triumph of scientific medicine. Sulfonamides, introduced in the 1930s, were the first selectively acting antibacterial agents that could be used in clinical medicine. Trimethoprim, on the other hand, came into clinical use at the end of the 1960s and can, with the exception of linezolid, be regarded as the last new antibacterial agent in the true sense.

The target of sulfonamides in bacteria is the enzyme dihydropteroate synthase. It is involved in the bacterial synthesis of the vital coenzyme, folic acid. Sulphonamides compete with the normal substrate of the enzyme, p-aminobenzoic acid, at the formation of dihydropteroate, which after the addition of glutamic acid gives folic acid. Mammalian cells lack dihydropteroate synthase and cannot synthesize folic acid. They are dependent on a nutritional supply of this vital compound. This is the basis for the selective action of sulfonamides on bacteria.

The first demonstration of the antibacterial effect of the chemically synthesized sulfonamides in experiments on animals was performed in 1932 by Gerhard Domagk at the University of Münster in Germany [2,3]. This can be regarded as the very first demonstration of the selective antibacterial action of a drug. Domagk’s work is now unjustly forgotten, but was widely known and highly valued by his international colleagues at the time, and in 1939 he was nominated for the Nobel Prize. At that time, the Nobel Prizes were severely discredited in the eyes of the Nazi regime in Germany, probably owing to the choice of Peace Prize laureates, and the Nazi government did not like to see any German as a nobelist. The Nobel committee of that time at the Karolinska Institute, with its chairman pathology professor Folke Henschen, was subjected to heavy pressure from the Nazi government, through its embassy in Stockholm and through its department of foreign affairs in Berlin, not to give the Nobel Prize to Domagk. The Nobel committee stood up to the pressure, however, and Domagk was announced as a laureate in October 1939. In his memoirs of 1957 Henschen, who knew Domagk personally, mentions that in the night following the announcement, Domagk was arrested by Nazi soldiers in his home in Wuppertal (Germany) and put in jail. The prison director on his round the next morning got irritated by Domagk’s behavior, and on his question Domagk answered “I am professor Domagk of the University of Münster”. “Weshalb sitzen Sie denn hier?” (Why are you here then?). Domagk’s reply: Ich habe den Nobelpreis bekommen” (I have been awarded the Nobel Prize). Domagk was not allowed to leave Germany for the award ceremony in Sweden. He would come to Stockholm only in 1947 to receive his Nobel diploma, but no prize money, as the will of Alfred Nobel clearly states that the money is forfeited should the laureate not come and participate in the award ceremony in Stockholm.

Chemically synthesized sulfonamides, with Domagk’s prontosil rubrum (metabolized to sulfanilamide in bacteria) as the first [3], have been widely used as inexpensive antibacterial agents against both Gram-positive and -negative pathogens. The great clinical interest in these drugs is reflected in the many derivatives that were synthesized. From a microbiologic point of view, they are all identical, but differ in pharmacokinetical properties. Sulfonamides are not used much nowadays for several reasons. The first reason is its side effects, which are quite common in treated patients. Adverse reactions from the skin and the hematopoietic system has led to restricted use. Sulfonamides seem to be the most commonly reported drugs causing all blood dyscrasias [4]. Systematic clinical studies have demonstrated that sulfonamides induced blood dyscrasias, including aplastic anemia, at a frequency of 5.3 per million defined daily doses of sulfonamides, with a fatality rate of 17% in the affected group [5]. Another reason for the limited use of sulfonamides was the rapid development of resistance after their introduction into clinical medicine. The detailed study of this resistance development regarding the oldest antibacterial agent in clinical use (i.e., sulfonamides) might be instructive for how to handle the occurrence of resistance to other antibacterials.

The simplest and most expected form of sulfonamide resistance would be a spontaneous mutation in the dihydropteroate synthase gene, resulting in a decreased affinity for its sulfonamide inhibitor. This is easily demonstrated experimentally in cultures of Escherichia coli, which when grown in the presence of sulphonamide, show spontaneous mutants with a 150-fold increase in the K_i value for the drug inhibiting the enzyme [6]. The mutated enzyme, however, also showed a tenfold increase in the K_m value for the normal substrate, p-aminobenzoic acid, resulting in a less efficient enzyme. This could be regarded as a trade-off for acquiring resistance in the evolutionary optimized enzyme. Clinically, this ought to result in unstable resistance, selected against in the absence of the drug. However, this does not seem to be the case in the clinical world of pathogens, where sulfonamide resistance has prevailed for decades in spite of a very low prevalence or absence of sulfonamides.

Sulfonamide resistance is commonly found in clinical isolates of Campylobacter jejuni. In this bacterium, it is mediated by four amino acid mutations in the dihydropteroate synthase gene to make the resistance enzyme differ from the sensitivity enzyme, and to give a 1000-fold increase in the K_i value for sulfonamide [7].

In Streptococcus pneumoniae, sulfonamide resistance is mediated by a different kind of chromosomal change, that is, by a six-nucleotide repeat mediating the repeat of two amino acids in the dihydropteroate synthase molecule. These changes occur at different locations in different clinical isolates, indicating that changes to sulfonamide resistance occurred independently on many occasions [8]. The extension of the enzyme protein by two amino acids would significantly alter its tertiary structure.

The very first experiments by Domagk on the antibacterial effect of sulfonamide were performed with the pathogen Streptococcus pyogenes almost 80 years ago [2], and the effects shown allowed sulfonamides to be much appreciated and frequently used as medicines in many bacterial infections, including those with S. pyogenes. They were also used for prophylaxis against streptococcal infections among soldiers in military training camps during World War II. Failures of this prophylaxis were observed, and were found to be due to the appearance of resistant streptococcal strains [9]. At that time, penicillin was introduced and soon dominated streptococcal treatment, which meant that the mentioned resistance was not characterized until later years and then only due to a general interest in resistance evolution [10,11]. At present, sulfonamide-resistant strains of S. pyogenes also seem to be prevalent, despite the fact that the use of sulfonamides for systemic use has been very limited or nonexistent for decades. This is an interesting example of the irreversibility of antimicrobial drug resistance in the absence of the selecting effect of the drug. The drug-resistant strains do not seem to be at any disadvantage in competition with their drug-susceptible relatives. A generalization of this observation would have important consequences for the handling of antibacterial drugs. The sulfonamide resistance of S. pyogenes turned out to be very different from the simple and straightforward mechanism of mutational changes in the target enzyme described previously. When the nucleotide sequence of the streptococcal dihydropteroate synthase gene of resistant isolates was compared with that of the susceptible one, the differences were so large that they could not be due to accumulated mutations. The resistance gene must have come from an external source and been introduced by transformation or transduction. The dihydropteroate synthase gene of resistant isolates showed areas of foreign DNA in a mosaic fashion, when closely compared to that of susceptible isolates. The origin of the foreign DNA is unknown, but a detailed analysis of the corresponding amino acid sequence showed differences that are known to mediate sulfonamide resistance [10,11]. Also in Neisseria meningitidis, which causes bacterial meningitis and fatal septicemia, sulfonamide resistance has been found to be caused by the transfer of resistance-mediating DNA between Neisseria strains. Sulfonamides were used extensively for the prophylaxis and treatment of meningococcal disease during the 1930s and 1940s. Today, sulfonamide resistance is commonly observed in clinical isolates of pathogenic N. meningitidis, which is another illustration of resistance stability in the absence of selection by the corresponding drug. There is also a possible association between pathogenicity and sulfonamide resistance, and between mortality rate and resistance [12]. Astonishingly large differences were found in the structure of the sulfonamide target gene and the dihydropteroate synthase gene, from susceptible and resistant strains of N. meningitidis, respectively [12,13]. Two classes of different resistance determinants were found among clinical isolates. In one class, the dihydropteroate synthase gene differed by approximately 10% between resistant and susceptible strains. This should mean that resistant strains had formed by recombination with horizontally transferred DNA rather than by the accumulation of mutations. Closer analysis showed mosaic genes, in which the central part was foreign while the outer parts were identical to those of susceptible isolates [12]. The origin of this resistance-mediating central part is probably other Neisseria species. This interpetation is supported by the finding of an 80-bp fragment identical to the corresponding part of the dihydropteroate synthase gene in Neisseria gonorrhoeae in one of the susceptible strains of N. meningitidis. This illustrates an exchange of genetic material between Neisseria species [14].

The other mentioned class of sulphonamide-resistant N. meningitidis strains showed a lower degree of difference to susceptible isolates in their dihydropteroate synthase target gene [14]. Several of these sulphonamide resistance genes were identical but distinct from the corresponding susceptibility genes. This indicates a horizontal transfer of genes followed by recombination. A closer study of the amino acid differences between resistance and susceptibility genes defined changes in three particular amino acids, among those that are conserved in all known dihydropteroate synthases [14]. When these amino acids were experimentally changed by site-directed mutagenesis to those of the consensus sequence of the susceptible enzyme, the K_i for sulfonamide dropped in the resulting enzyme [15]. There were also changes in the K_m value for the ordinary substrate p-aminobenzoic acid, which, taken together with the resistance changes, were interpreted to point to the existence of other amino acid changes compensating for the possible detrimental effect on enzyme efficiency of the resistance-mediating amino acid changes [15]. This interpretation, inferring the existence of mutations adapting the enzyme to optimal function in the presence of resistance mutations, could explain the stability of sulfonamide resistance in N. meningitidis, allowing it to exist and be prevalent among strains isolated today, in spite of the fact that sulfonamides have not been used systemically for decades. This resistance could then be looked at as a microbiological scar from earlier drug use for the treatment and prophylaxis of bacterial meningitis.

Plasmid-borne extrachromosomal resistance to sulfonamides has also been frequently observed among Gram-negative enterobacteria. Astonishingly, this resistance was found to be mediated by plasmid-borne genes expressing highly sulphonamide-resistant dihydropteroate synthase, making the bacterium diploid of sorts, carrying two genes for dihydropteroate synthase: one chromosomal gene expressing the normal dihydropteroate synthase of the bacterium, which will be incapacitated in the presence of sulfonamide, and one plasmid-borne gene producing a dihydropteroate synthase, which is insensitive to the drug [16,17]. Three genes expressing resistant enzymes of this type are known and characterized [18,19] – their origins are unknown. These resistance enzymes are remarkable in their ability to discriminate between the normal substrate, p-aminobenzoic acid, and the structurally very similar sulfonamide. The normal substrate binds well, showing low K_m values, while the sulfonamide inhibitor is almost without an inhibiting effect, also at very high concentrations.

Resistance to trimethoprim

The target for the antibacterial action of trimethoprim is the bacterial enzyme dihydrofolate reductase. Trimethoprim can be regarded as an antifolate, inhibiting dihydrofolate reductase by competing with its normal substrate, dihydrofolate [20]. It is therefore, as a drug, related to sulfonamides in the sense that it interferes with bacterial folate metabolism. Trimethoprim acts selectively on bacterial dihydrofolate reductases leaving mammalian enzymes untouched, which allows its use as an antibacterial drug. There is a structural explanation for this, in that x-ray crystallography studies have shown that trimethoprim does not fit into the nucleotide-binding site of the mammalian enzyme, while the corresponding site of bacterial enzymes readily accepts it [21]. Trimethoprim has a broad antibacterial spectrum, and since it attacks a later step in the same enzymic pathway as sulfonamides, they act synergistically. This has been successfully exploited in the combination drug co-trimoxazole.

Some bacteria, such as Campylobacter jejuni and Helicobacter pylori, seem to be naturally resistant to trimethoprim. These bacteria lack a chromosomal gene for dihydrofolate reductase and thus do not offer any target for the drug [22]. Otherwise, chromosomal resistance to trimethoprim has been observed in several pathogenic bacteria. One example is a clinical isolate of Escherichia coli, which overproduced its chromosomal dihydrofolate reductase several 100-fold, by a combination of several types of mutations, which increased the enzyme gene expression by increasing promoter efficiency, optimizing ribosome binding and resulting in more efficient codon usage [23].

Chromosomal resistance to trimethoprim in S. pneumoniae is rather common. Detailed studies on the nucleotide sequence of its chromosomal dihydrofolate reductase gene revealed a number of changes so extensive that a horizontal transfer of resistance genes was indicated. This interpretation was experimentally supported by the ability of chromosomal DNA and PCR products from resistant strains to transform a susceptible strain to resistance [24].

Plasmid-borne resistance to trimethoprim is common. In parallel to that of sulfonamides, it is mediated by foreign resistant variations of the trimethoprim target enzyme, dihydrofolate reductase. The first of these was found decades ago [25], and since then new ones have continually been added to the list, where now at least 30 different resistance genes have been found. The proteins expressed from these genes are numbered continuously from the first one found. The precise origin is not known in any case. The first found, dfr1, seems to be the most common. It occurs in a cassette on both class 1 and 2 integrons [20]. Transposon Tn7 has spread very suceessfully, mainly owing to its frequent insertion into a preferred site on the chromosome of E. coli and many other enterobacteria. It carries a class 2 integron mediating a dfr1 cassette [20]. Among the horizontally moving plasmid-borne trimethoprim resistance genes, four (dfr2a, dfr2b, dfr2c and dfr2d) are closely related between themselves, but are so different from other trimethoprim resistance genes that they could not be included in the phylogenetic tree (Figure 1), where the relationship between the other trimethoprim-resistance genes could be demonstrated and compared. Their enzymically active forms appear as tetramers and the monomer polypeptides consist of 78 amino acids, which are 67% identical between the four enzymes.

The active tetramers show dihydrofolate activity, which is almost insensitive to trimethoprim, making hosts so drug resistant that colonies of them can be observed growing among trimethoprim crystals on an agar plate saturated with the drug [20,26]. The phylogenetic tree in Figure 1 relates different dihydrofolate reductases on the basis of amino acid sequence alignment and parsimony analysis [27]. In this tree, the trimethoprim-resistance enzymes dfr1, dfr5, dfr6, dfr7 and dfr14 form a rather closely related group of peptides. Otherwise, the trimethoprim resistance enzymes are scattered all over the tree. This is consistent with the idea that the resistance enzymes have their origin in a variety of different organisms. dfr3, however, is closely related to the chromosomal enzyme of enterobacteria, which might provide clues to its origin. Furthermore, the drug-insensitive dihydrofolate reductase, S1, borne on the frequently occurring transposon Tn4003[28,29], is almost identical to the chromosomal enzyme of Staphylococcus epidermidis. It differs by only three amino acid substitutions, and it has been suggested that a mutated form of S. epidermidis has moved horizontally into other staphylococcal species [30].

One of the horizontally spread resistance enzymes, dfr9, only distantly related to the mentioned rather closely related group of five peptides (Figure 1), was originally found expressed from a gene on large transferable plasmids in isolates of E. coli from swine [31]. The corresponding gene was frequently found among swine isolates, which could be a reflection of the common veterinarian prescription of trimethoprim in swine rearing. It was, however, only very rarely found in isolates from patients [32]. This could reflect a situation where a resistance gene selected in an agricultural context is spreading into human pathogens [33]. A closer study showed the dfr9 gene to be located on a truncated transposon Tn5093, previously found on a plasmid in the plant pathogen Erwinia amylovora, causing fire blight on apple trees [34]. This transposon carries two streptomycin resistance genes, and it probably evolved under the selection pressure of streptomycin, ubiquitously used for the control of E. amylovora in many countries [35]. The dfr9 gene was found to be inserted in one of the streptomycin resistance genes at the right hand end of the truncated Tn5393. The occurrence of dfr9-mediating trimethoprim resistance in isolates of E. coli in Sweden, and located on a truncated form of a transposon originally observed to carry genes for streptomycin resistance in a plant pathogen in the USA, might be a powerful demonstration of the efficient horizontal spread of resistance genes in response to the heavy use of antibacterial agents in agriculture. Modern pig-rearing in large stables with hundreds of animals could be seen as large genetic laboratories with gigantic populations of genetically intercommunicating bacteria, also allowing very rare genetic events for mobilizing resistance genes to the surface under the selection pressure of antibiotics.

Conclusion

Trimethoprim and sulfonamides are inexpensive and efficient antibacterial agents that have suffered severely in clinical usefulness owing to resistance among pathogenic bacteria. The study of the molecular mechanisms of this resistance and its spread among pathogens mediates a representative picture of how antibacterial resistance evolves in general. A detailed knowledge of the manifold evolutionary solutions that nature has found for resistance development might be helpful in finding countermeasures, and perhaps for also providing the medicinal chemist with clues for the design of new antibacterial agents.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.