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Nucleotide sequence analysis of ORF1 from the integron on the broad-host-range plasmid R751 revealed that the first 94 of 110 codons of ORF1 from R751 are identical to ORF4, an open reading frame from the 3' conserved segment of other integrons found in gram-negative bacteria, after which point they diverged completely. The predicted products of both ORFI and ORF4 share homology with the multidrug exporter QacC. Phenotypic analysis revealed that ORFI specifies a resistance profile to antiseptics and disinfectants almost identical to that of qacC, whereas ORF4 specifies much lower levels of resistance to these compounds. ORF4, whose product lacks the C-terminal 16 amino acids of the ORF1 protein, may have evolved by the interruption of ORF1 from the insertion of a DNA segment carrying a sufl sulfonamide resistance determinant. Hence, ORF1 was designated qacE, and its partially functional deletion derivative, ORF4, was designated qacEAI. Fluorimetric experiments indicated that the mechanism of resistance mediated by QacE, the protein specified by qacE, is active export energized by proton motive force. Amino acid sequence comparisons revealed that QacE is related to a family of small multidrug export proteins with four transmembrane segments.

A 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 containing 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 30,126 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.

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Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrasemediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn2l and Tn5O86. Several characteristics suggest that this element is a transposon, which we call TnSO9O. TnS090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins.

BackgroundPrompt and effective malaria diagnosis not only alleviates individual suffering, but also decreases malaria transmission at the community level. The commonly used diagnostic methods, microscopy and rapid diagnostic tests, are usually insensitive at very low-density parasitaemia. Molecular techniques, on the other hand, allow the detection of low-level, sub-microscopic parasitaemia. This study aimed to explore the presence of sub-microscopic Plasmodium falciparum infections using polymerase chain reaction (PCR). The PCR-based parasite prevalence was compared against microscopy and rapid diagnostic test (RDT).MethodsThis study used 1,453 blood samples collected from clinical patients and sub-clinical subjects to determine the prevalence of sub-microscopic P. falciparum carriages. Subsets of RDT and microscopy negative blood samples were tested by PCR while all RDT and microscopically confirmed P. falciparum-infected samples were subjected to PCR. Finger-prick blood samples spotted on filter paper were used for parasite genomic DNA extraction.ResultsThe prevalence of sub-microscopic P. falciparum carriage was 19.2% (77/400) (95% CI = 15. 4–23.1). Microscopy-based prevalence of P. falciparum infection was 3.7% (54/1,453) while the prevalence was 6.9% (100/1,453) using RDT alone. Using microscopy and PCR, the estimated parasite prevalence was 20.6% if PCR were performed in 1,453 blood samples. The prevalence was estimated to be 22.7% if RDT and PCR were used. Of 54 microscopically confirmed P. falciparum-infected subjects, PCR detected 90.7% (49/54). Out of 100 RDT-confirmed P. falciparum infections; PCR detected 80.0% (80/100). The sensitivity of PCR relative to microscopy and RDT was, therefore, 90.7% and 80%, respectively. The sensitivity of microscopy and RDT relative to PCR was 16.5 (49/299) and 24.2% (80/330), respectively. The overall PCR-based prevalence of P. falciparum infection was 5.6- and 3.3 fold higher than that determined by microscopy and RDT, respectively. None of the sub-microscopic subjects had severe anaemia, though 29.4% had mild anaemia (10–11.9 g/dl).ConclusionsAsymptomatic, low-density malaria infection was common in the study area and PCR may be a better tool for measuring Plasmodium prevalence than microscopy and RDT. The inadequate sensitivity of the diagnostic methods to detect substantial number of sub-microscopic parasitaemia would undoubtedly affect malaria control efforts, making reduction of transmission more difficult. RDT and microscopy-based prevalence studies and subsequent reports of reduction in malaria incidence underestimate the true pictures of P. falciparum infections in the community. PCR, on the other hand, seems to have reasonable sensitivity to detect a higher number of infected subjects with low and sub-microscopic parasite densities than RDTs or microscopy.

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-...

This paper describes the second type of sulfonamide resistance gene, sullI, and its surroundings in three plasmids, pGS03B, RSF1010, and pGS05. pGS03B3 is identical to pBP1 (36), a 6.3-kilobase-pair (kb) multicopy plasmid that has a resistance region homologous to that of the IncQ plasmid RSF1010, and carries linked sulfonamide and streptomycin resistance (39). The streptomycin resistance specified by these plasmids is due to an aminoglycoside phosphotransferase APH-(3") (21). Plasmid RSF1010 (8.7 kb) is a broad-host-range plasmid found in many different gramnegative bacteria, including the enterobacteria and Pseudomonas aeruginosa (3,11,16). Many IncQ plasmids have only minor differences in the antibiotic resistance region, and it has been suggested that a single IncQ plasmid has spread efficiently around the world with minor modifications (6, 15). The third plasmid, pGS05, is a 100-kb conjugative plasmid specifying the sulfonamide and streptomycin resis-* Corresponding author. tance found in a clinical isolate of Escherichia coli. The sulfonamide resistance DHPS of pGS05 is of type II (37).The nucleotide sequence of the gene coding for the type II sulfonamide resistance DHPS of RSF1010 and pGS05 was determined and compared with the sequence of the sulI gene. The two types of plasmid-encoded sulfonamide resistance enzymes were also compared with the chromosomal DHPS from a sulfonamide-resistant mutant of Streptococcus pneumoniae (24). The polypeptides expressed by the gramnegative sul genes were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with the maxicell system. MATERIALS AND METHODSBacterial strains and plasmids. E. coli C600 (F-lac leu supE thi thr tonA) was used as host for vector pBR322 (2), JM83 [ara A(lac-proAB)