No 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-...

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pM sensitivity each consuming only 1 l of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibodybased probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.

The nucleotide sequences of the chromosomal dihydropteroate synthase (dhps) genes in sulfonamidesusceptible 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 resistancemediating 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.

A sensitive PCR assay that detects the thermophilic campylobactersC. jejuni, C. coli, C. lari, andC. upsaliensis is reported. Furthermore, by digestion of the PCR products with two restriction enzymes, species differentiation was demonstrated. Thus, the present method has the potential to be used for both detection and identification of thermophilicCampylobacter species.