Ola Sköld

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.

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.

NON-PALINDROMIC ATTL SITES OF INTEGRONS ARE CAPABLE OF SITE-SPECIFIC RECOMBINATION WITH ONE ANOTHER AND WITH SECONDARY TARGETS

Genes borne on cassettes are mobile owing to site‐specific recombination systems called integrons, which have created various combinations of antibiotic resistance genes in R‐plasmids. In these processes, the palindromic site, attC (59‐base element), at cassette junctions has been proposed as being essential. Excised and circularized cassettes have been found to integrate with preference for an attI site at one end of the conserved sequence in integrons. In this work, we give evidence that recombination is possible in the absence of the highly organized attC sites between the more simply organized attI sites. Furthermore, at a very low frequency representing the background in our recombination assay, we observed cross‐overs between attI and secondary sites. To characterize recombination excluding the attC sites, we have used naturally occurring attI variants and constructed mutants. The cross‐over point was identified between a guanine and a thymine in attI using point mutations. Progressive deletions showed the extent of attI and identified two important regions in the conserved sequence 5′ of the cross‐over point. A region 27–36 bp 5′ of attI influenced recombination with attC sites only, whereas a sequence 9–14 bp 5′ of the cross‐over point in attI was important for recombination with both attI and attC. Recombination between attI and secondary sites could allow fusion of the conserved sequence encoding the integron site‐specific recombinase to new sequences.

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

R-Factor-Mediated Resistance to Sulfonamides by a Plasmid-Borne, Drug-Resistant Dihydropteroate Synthase

Evidence was found for the existence of an episome-specified variant of the enzyme dihydropteroate synthase involved in folic acid formation. Since the plasmid-borne enzyme showed a decreased susceptibility for sulfonamide inhibition and was transferable together with resistance to this drug, it is proposed that diploidy for the target enzyme in some cases could be the mechanism of R-factor-mediated resistance to sulfonamides. Two types of evidence were obtained. One was the rescue from temperature sensitivity of bacterial mutants with a lesion in the chromosomal dihydropteroate synthase by the R factor R1dr19 mediating sulfonamide resistance. The other evidence was found by the determination of dihydropteroate forming activity in extracts from R− and R+ bacteria. Cells harboring R1dr19 were found to contain an enzyme activity which was far less susceptible to sulfonamide inhibition than the corresponding activity from R− cells.

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.

Complete Sequence of a β-Lactamase-Encoding Plasmid in Neisseria meningitidis

ABSTRACT Identical β-lactamase-encoding (TEM-1) plasmids were found in two different clinical Neisseria meningitidis strains. They were completely sequenced (5,597 bp) and designated pAB6. The plasmid is almost identical to Neisseria gonorrhoeae plasmid pJD5 (5,599 kb) and may have been picked up from a gonococcus in vivo.

Trimethoprim resistance plasmids of different origin encode different drug-resistant dihydrofolate reductases

Abstract Several plasmids mediating resistance to folic acid analogs were studied. The plasmids were in part newly isolated from clinical material and in part R factors studied earlier, such as R483, R721, R751, and R388. By gel chromatography, plasmid-carrying bacterial strains were all found to produce drug-resistant dihydrofolate reductases of a molecular weight distinctly larger than that of the chromosomal enzyme of the host. By gel electrophoresis and zymographic detection technique, analog inhibition characteristics, heat sensitivity, and pH optimum curves, the dihydrofolate reductases induced by R483, R751, and R388, respectively, could be clearly discerned as separate enzymes. Of the newly isolated plasmids all but one coded for a dihydrofolate reductase similar to that of R483. The aberrant one seemed to yield a new enzyme variant as judged from its drug inhibition characteristics and its pH optimum profile. Large differences in drug insensitivity were observed, thus the R751 and R388 enzymes were virtually insensitive to folic acid analogs, whereas the corresponding enzymes from the newly isolated plasmids, and from R483 showed a substantially higher sensitivity. On the other hand these latter enzymes were overproduced, in that the plasmid-carrying bacteria showed a 10- to 20-fold higher content of dihydrofolate reductase than the plasmid-free host strain. Among newly isolated trimethoprim-resistant strains, one was found which overproduced dihydrofolate reductase about 200-fold. In this case the enzyme was only slightly more resistant to folic acid analogs than the chromosomal Escherichia coli K-12 enzyme, and did not seem to be plasmid borne.

An Integron Cassette Carrying dfr1 with 90-bp Repeat Sequences Located on the Chromosome of Trimethoprim-Resistant Isolates of Campylobacter jejuni

The frequent occurrence of high-level trimethoprim resistance in clinical isolates of Campylobacter jejuni was shown to be related to the acquisition of foreign resistance genes (dfrl or dfr9 or both) coding for resistant variants of the enzyme dihydrofolate reductase, the target of trimethoprim. The dfr1 gene detected on the chromosome of 40 different clinical strains of C. jejuni was studied further regarding structure and genetic organization. Most of the dfr1 genes were found as integron cassettes inserted in the chromosome. In 36% of the examined isolated, the dfr1 gene showed identity to that previously characterized in trimethoprim-resistant Escherichia coli. In 40% of the cases, however, a variant of the dfr1 gene containing a 90-bp direct repeat was detected, and in 5% of the isolates, the repeat-containing dfr1 variant was found to occur in the form of two cassettes in tandem in an integron context. The existence of the 90-bp repeat within the coding sequence of the dfr1gene was found to play a role in the adaptation of C. jejuni to ambient concentrations of trimethoprim.

Spread of a newly found trimethoprim resistance gene, dhfrIX, among porcine isolates and human pathogens.

A plasmid-borne gene mediating trimethoprim resistance, dhfrIX, newly found among porcine strains of Escherichia coli, was observed at a frequency of 11% among trimethoprim-resistant veterinary isolates. This rather high frequency of dhfrIX could be due to the extensive use of trimethoprim in veterinary practice in Sweden. After searching several hundred clinical isolates, one human E. coli strain was also found to harbor the dhfrIX gene. Thus, the dhfrIX gene seems to have spread from porcine bacteria to human pathogens. Furthermore, the occurrence of other genes coding for resistant dihydrofolate reductase enzymes (dhfrI, dhfrII, dhfrV, dhfrVII, and dhfrVIII) among the porcine isolates was investigated. In addition, association of dhfr genes with the integraselike open reading frames of transposons Tn7 and Tn21 was studied. In colony hybridization experiments, both dhfrI and dhfrII were found associated with these integrase genes. The most common combination was dhfrI and int-Tn7, indicating a high prevalence of Tn7.