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

Abstract

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.Sulfonamides have played a very important part in antibacterial chemotherapy for 6 decades. Their target is the enzyme dihydropteroate synthase (DHPS) (EC 2.5.1.15), which catalyzes the formation of dihydropteroic acid in bacterial and some eucaryotic cells, but it is not present in human cells. This difference is the basis for the selective action of sulfonamide drugs, which act as competitive inhibitors of DHPS, thereby blocking folate biosynthesis in the bacterial cell (5). Sulfonamides are structural analogs to the normal substrate p-aminobenzoic acid and can indeed function as alternative substrates to produce a sulfa-containing pteroate analog (16,22). Chromosomal mutations in the dhps gene resulting in low levels of sulfonamide resistance (Ͻ0.05 mM) can be isolated in the laboratory (13,19), and naturally occurring resistance to high concentrations of sulfonamides (Ͼ0.5 mM) has been observed in gram-negative, enteric bacteria. This resistance is plasmid-borne and due to the presence of alternative drugresistant variants of the enzyme (23). Two such plasmid-encoded enzymes have been detected, and the nucleotide sequences of the corresponding genes have been determined (15,21,24).In Neisseria meningitidis, traits mediating high resistance (Ն0.5 mM) to sulfonamides were found to be located chromosomally (10,14). Most of the sulfonamide-resistant clinical strains of N. meningitidis were found to contain a dhps gene that was about 10% different from the corresponding gene in sulfonamide-sensitive isolates. From this large difference it was concluded that the resistance had been introduced by recombination rather than by mutation. One characteristic of these chromosomal resistance genes is an insertion of 6 bp, creating two extra codons in the sequence. Two sulfonamide-resistant strains of N. meningitidis differed in their sequences from this often-found form of resistance by lacking the 6-...

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

“…Standard procedures (25) were used for ligations, restriction enzyme digestions, and agarose gel electrophoresis. The dhps gene in the sulfonamideresistant meningococcus 5M, isolated in Slovenia (14), was PCR amplified with primers NM1 and NM2 and DNA sequenced as described previously (10). The mutagenesis procedure of Vandeyar et al (30) was followed as described before (10).…”

“…The 0.33-kb BamHI-PstI fragment of the dhps gene of E. coli and the 1.25-kb kanamycin resistance (Km r ) gene cartridge (Pharmacia, Uppsala, Sweden) were used as probes. The entire dhps gene, PCR amplified with primers EC2 and EC3 as described previously (10), was used to probe the Kohara miniset phage collection (13), kindly provided by Carin Karlsson, Department of Microbiology, Uppsala University, Uppsala, Sweden. The probes were labeled with [␣-32 P]dCTP from New England Nuclear by the random priming method.…”

“…Resistance has been shown to be mediated by altered forms of the chromosomal dhps gene (10,21). Two types of resistant DHPSs have so far been identified by nucleotide sequence comparisons.…”

“…One type can be considered to be encoded by a meningococcal wild-type gene that has mutated to mediate resistance, while the gene encoding the other type differs substantially (10%) from the corresponding genes in sulfonamidesusceptible strains of N. meningitidis. It was thus concluded that the latter type of resistance determinant has spread by horizontal transfer from another bacterium (10,21). Both types of resistance determinants have been analyzed by sitedirected mutagenesis, and differences from the wild-type enzymes responsible for resistance have been identified (10).…”

“…It was thus concluded that the latter type of resistance determinant has spread by horizontal transfer from another bacterium (10,21). Both types of resistance determinants have been analyzed by sitedirected mutagenesis, and differences from the wild-type enzymes responsible for resistance have been identified (10).…”