Chromosomal location of antibiotic resistance genes in Neisseria gonorrhoeae.

Transformation with purified plasmid and chromosomal deoxyribonucleic acid from a clinical isolate of Neisseria gonorrhoeae showed that each of seven loci affecting drug resistance (penA, penB, ery, str, tet, chl, and env) was chromosomal.     

淋球菌耐药性与耐药质粒的研究

为了解湛江地区淋球菌耐药现状及耐药质粒的分布,并初步探讨其相互关系,对１９９８￣１９９９年广东省湛江地区健康鉴定出的９８株淋球菌流行株,应用纸片扩散法测定细胞对１０种抗生素的敏感性,并采用碱裂解法进行质粒抽提,分析耐药质粒的分布情况,选取ＮＧ４、ＮＧ３１、ＮＧ４３、ＮＧ７０进行细菌的接合试验,以观察耐药质粒能否通过接合进行传递以及传递后受体菌耐药性的变化。结果显示６．１２％菌株对全部抗生素敏感,４８．９６％的菌株对３种及３种以上的抗生素耐药;共检出四种不同分子量的质闰,７．４ｋｂ和４．２ｋｂ质粒检出率较高,分别为５９．１６％和６７．３２％,质粒谱型１２种,以７．４ｋｇ＋４．２ｋｇ和３９．５ｋｇ＋７．４ｋｇ＋４．２ｋｇ为主,占３８．７６％。在细菌的接合传递试验中,ＮＧ４３可通过接合传递将其耐药性传递给淋球菌及大肠     

Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene.

The hygromycin phosphotransferase gene was fused in-frame with the herpes simplex virus type 1 thymidine kinase gene. The resulting fusion gene (termed HyTK) confers hygromycin B resistance for dominant positive selection and ganciclovir sensitivity for negative selection and provides a means by which these selectable phenotypes may be expressed and regulated as a single genetic entity.     

Isolation and analysis of a fur mutant of Neisseria gonorrhoeae.

The pathogenic Neisseria spp. produce a number of iron-regulated gene products that are thought to be important in virulence. Iron-responsive regulation of these gene products has been attributed to the presence in Neisseria spp. of the Fur (ferric uptake regulation) protein. Evidence for the role of Fur in neisserial iron regulation has been indirect because of the inability to make fur null mutations. To circumvent this problem, we used manganese selection to isolate missense mutations of Neisseria gonorrhoeae fur. We show that a mutation in gonococcal fur resulted in reduced modulation of expression of four well-studied iron-repressed genes and affected the iron regulation of a broad range of other genes as judged by two-dimensional polyacrylamide gel electrophoresis (PAGE). All 15 of the iron-repressed spots observed by two-dimensional PAGE were at least partially derepressed in the fur mutant, and 17 of the 45 iron-induced spots were affected by the fur mutation. Thus, Fur plays a central role in regulation of iron-repressed gonococcal genes and appears to be involved in regulation of many iron-induced genes. The size and complexity of the iron regulons in N. gonorrhoeae are much greater than previously recognized.     

Loss of low-level antibiotic resistance in Neisseria gonorrhoeae due to env mutations.

Mutations (env) which resulted in increased sensitivity of gonococci to diverse compounds were studied by transformation. Strains carrying an env mutation were more sensitive than wild-type strains to several antibiotics, dyes, and detergents. The env mutations resulted in complete phenotypic suppression of low-level resistance to these same drugs determined by mutation at ery. Recombination was observed in transformation crosses between various env mutants. The env locus was not linked to the cluster of antibiotic resistance genes near str and spc.     

Genetic analysis of drug resistance in Neisseria gonorrhoeae: production of increased resistance by the combination of two antibiotic resistance loci.

The studies reported here demonstrate that increased resistance of Neisseria gonorrhoeae to penicillin, tetracycline, and chloramphenicol results from the combined effect of two resistance loci. As shown by experiments with deoxyribonucleic acid from transformants carrying only a single resistance locus, transformants with an incresed level of resistance to penicillin result from the combination of a penicillin-specific locus, pen, and a multiple resistance locus, mtr. Similarly, transformants with an increased level of resistance to tetracycline result from the combination of mtr and a tetracycline-specific locus, tet. Transformants with an increased level of resistance to chloramphenicol result from the combination of mtr and a chloramphenicol-specific locus, cml. Deoxyribonucleic acid dilution experiments established that only a single dose of each of the two required resistance loci is necessary to give higher-level resistance. Higher-level-resistant transformants were not obtained when a double dose of one resistance locus or a combination of loci pairs other than mtr and pen, mtr and tet, or mtr and cml was introduced into a recipient. Combinations of the mtr and tet genes resulted in increased resistance to semisynthetic tetracyclines. The presence of the mtr and pen genes resulted in increased resistance to penicillinase-stable penicillins.     

Influence of conserved and hypervariable genetic markers on genotyping circulating strains of Neisseria gonorrhoeae

Presently there is no vaccine against Neisseria gonorrhoeae and therefore accurate information on gonococcal transmission plays a crucial role for interventions designed to limit the spread of infections caused by this microorganism. We evaluated the impact of two different categories of genetic markers, (i) concatenated sequences of 10 housekeeping genes and (ii) hypervariable porB DNA sequences, on the genetic relatedness and subsequently on genotyping analysis of this human pathogen. Eighty gonococcal isolates from Canada, China, the US, Argentina, Venezuela and Chile, collected over different times, were analyzed. Our results show that the choice of genetic marker had a profound effect on the interpretation of genotyping results associated with N. gonorrhoeae. The concatenated sequences of the housekeeping genes preserved the genetic relatedness of closely related isolates, enabling detection of the predominant strains circulating within a community (Saskatchewan, Canada) over an extended period of time. In contrast, a genetic marker based on antigen gene, porB, may lead to a failure to detect these predominant circulating strains. Based on the analysis of the DNA sequences of the 10 housekeeping genes, we identified two major clonal complexes, CC33 and CC22, which comprised STs from China, and Argentina as well as two STs from Canada. Several minor clonal complexes were observed among isolates from Saskatchewan. eBURST analysis suggested that the majority of the tested gonococcal isolates from Saskatchewan, Canada were endemic, with only a couple of genotypes introduced.     

126株淋病奈瑟菌耐药性分析及其所致感染的中西医结合治疗

目的研究近几年来淋病奈瑟菌(简称淋球菌)对抗生素的敏感性,探讨中西医结合治疗的有效性。方法对我科从2000年1月～2007年6月收治保存的126株淋球菌分别用纸片扩散法和快速药敏测试法进行15种药物的药敏检测,并用头孢曲松合并中药治疗。结果用快速定量药敏检测法测定的最小抑菌浓度(MIC)与用K-B法基本相符。对环丙沙星的耐药率为40%,1.50%的菌株耐大观霉素(MIC≥64μg/ml);所有菌株均对头孢曲松敏感。中西医结合治疗所有病例均获得痊愈。结论头孢曲松药物敏感性无明显变化,6年间未发现耐药菌株;环丙沙星耐药性显著上升。中西医结合在淋病治疗中有效可行。     

Type IV prepilin peptidase gene of Neisseria gonorrhoeae MS11: presence of a related gene in other piliated and nonpiliated Neisseria strains.

The assembly of type IV pili in Neisseria gonorrhoeae is a complex process likely to require the products of many genes. One of these is the enzyme prepilin peptidase, which cleaves and then N methylates the precursor pilin subunits prior to their assembly into pili. We have used a PCR amplification strategy to clone the N. gonorrhoeae prepilin peptidase gene, pilDNg. A single copy of the gene is shown to be present in the chromosome. Its product promotes correct cleavage of the gonococcal prepillin in Escherichia coli cells carrying both the prepilin peptidase gene and the pilin structural gene. PilDNg also cleaves prePulG, a type IV pilin-like protein of Klebsiella oxytoca. Moreover, PilDNg complements a mutation in the gene coding for the prepilin peptidase-like protein of K. oxytoca, pulO, partially restoring PulG-PulO-dependent extracellular secretion of the enzyme pullulanase. Finally, we show that genes homologous to pilDNg are present and expressed in a variety of species in the genus Neisseria, including some commensal strains.     

Porin-mediated antibiotic resistance in Neisseria gonorrhoeae: ion, solute, and antibiotic permeation through PIB proteins with penB mutations

Neisseria gonorrhoeae has two porins, PIA and PIB, whose genes (porA and porB, respectively) are alleles of a single por locus. We recently demonstrated that penB mutations at positions 120 and 121 in PIB, which are presumed to reside in loop 3 that forms the pore constriction zone, confer intermediate-level resistance to penicillin and tetracycline (M. Olesky, M. Hobbs, and R. A. Nicholas, Antimicrob. Agents Chemother. 46:2811-2820, 2002). In the present study, we investigated the electrophysiological properties as well as solute and antibiotic permeation rates of recombinant PIB proteins containing penB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H). In planar lipid bilayers, the predominant conducting state of each porin variant was 30 to 40% of the wild type, even though the anion selectivity and maximum channel conductance of each PIB variant was similar to that of the wild type. Liposome-swelling experiments revealed no significant differences in the permeation of sugars or beta-lactam antibiotics through the wild type or PIB variants. Although these results are seemingly contradictory with the ability of these variants to increase antibiotic resistance, they are consistent with MIC data showing that these porin mutations confer resistance only in strains containing an mtrR mutation, which increases expression of the MtrC-MtrD-MtrE efflux pump. Moreover, both the mtrR and penB mutations were required to decrease in vivo permeation rates below those observed in the parental strain containing either mtrR or porin mutations alone. Thus, these data demonstrate a novel mechanism of porin-mediated resistance in which mutations in PIB have no affect on antibiotic permeation alone but instead act synergistically with the MtrC-MtrD-MtrE efflux pump in the development of antibiotic resistance in gonococci.     

Cotransformation of temperature sensitivity and nutritional markers in Neisseria gonorrhoeae.

Cotransformation remains the only tool for establishing linkage in Neisseria gonorrhoeae. Because of the difficulty of inducing auxotrophic markers via mutagenesis in this species, most previous studies have utilized antibiotic resistance and naturally occurring (auxotypic) auxotrophic markers. We have succeeded in isolating auxotrophic and temperature-sensitive mutants. The temperature-sensitive mutants have been characterized by their growth on complex and defined media at 31, 37, and 40 degrees C. Two of the mutants exhibited an unusual pattern of temperature sensitivity--growth on the defined medium but absence of growth on the complex medium at 37 degrees C. Both mutants, however, were temperature-sensitive on the two media at 40 degrees C. We have demonstrated linkages between markers isolated in our laboratory and the auxotypic markers of the clinical isolate RUG208. Ts-2 exhibited 85 to 95% linkage to Arg- and his-2 exhibited 40% linkage to Val-. In addition weak linkages were shown between his-2 and Arg- (2 to 6%) and between Arg- and Val- (3 to 5%). Linkages among his-2, Arg-, and Val- which could be demonstrated when deoxyribonucleic acid from strain F62 was used to transform RUG208 were absent when F62 was used as recipient for RUG208 DNA. Our data are consistent with a tentative map order of his-2, Val-, Arg-, Ts-2.     

A recombinase-mediated system for elimination of antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains.

A TnpI-mediated site-specific recombination system to construct genetically modified Bacillus thuringiensis strains was developed. Recombinant B. thuringiensis strains from which antibiotic resistance genes can be selectively eliminated were obtained in vivo with a new vector based on the specific resolution site of transposon Tn4430. For example, a cryIC gene, whose product is active against Spodoptera littoralis, was introduced into B. thuringiensis Kto harboring a cryIA(c) gene active against Ostrinia nubilalis. The resulting strain had a broader activity spectrum than that of the parental strain. It contained only B. thuringiensis DNA and was free of antibiotic resistance genes. This should facilitate regulatory approval for its development as a commercial biopesticide.     

Crystal structures of penicillin-binding protein 2 from penicillin-susceptible and -resistant strains of Neisseria gonorrhoeae reveal an unexpectedly subtle mechanism for antibiotic resistance

Penicillin-binding protein 2 (PBP2) from N. gonorrhoeae is the major molecular target for beta-lactam antibiotics used to treat gonococcal infections. PBP2 from penicillin-resistant strains of N. gonorrhoeae harbors an aspartate insertion after position 345 (Asp-345a) and 4-8 additional mutations, but how these alter the architecture of the protein is unknown. We have determined the crystal structure of PBP2 derived from the penicillin-susceptible strain FA19, which shows that the likely effect of Asp-345a is to alter a hydrogen-bonding network involving Asp-346 and the SXN triad at the active site. We have also solved the crystal structure of PBP2 derived from the penicillin-resistant strain FA6140 that contains four mutations near the C terminus of the protein. Although these mutations lower the second order rate of acylation for penicillin by 5-fold relative to wild type, comparison of the two structures shows only minor structural differences, with the positions of the conserved residues in the active site essentially the same in both. Kinetic analyses indicate that two mutations, P551S and F504L, are mainly responsible for the decrease in acylation rate. Melting curves show that the four mutations lower the thermal stability of the enzyme. Overall, these data suggest that the molecular mechanism underlying antibiotic resistance contributed by the four mutations is subtle and involves a small but measurable disordering of residues in the active site region that either restricts the binding of antibiotic or impedes conformational changes that are required for acylation by beta-lactam antibiotics.     

Microarray genomotyping of key experimental strains of Neisseria gonorrhoeae reveals gene complement diversity and five new neisserial genes associated with Minimal Mobile Elements.

Background  

There are four widely used experimental strains of N. gonorrhoeae, one of which has been sequenced and used as the basis for the construction of a multi-strain, mutli-species pan-neisserial microarray. Although the N. gonorrhoeae population structure is thought to be less diverse than N. meningitidis, there are some recognized gene-complement differences between strains, including the 59 genes of the Gonococcal Genetic Island. In this study we have investigated the three experimental strains that have not been sequenced to determine the extent and nature of their similarities and differences.     

Biological properties of clinical staphylococcal strains with a varying number of antibiotic resistance markers]

The study of a number of biological properties of 1881 clinical strains of Staphylococcus showed that in the group of the antibiotic resistant staphylococci there was a tendency for different manifestation of some biological properties depending on the number of the resistance determinants. The staphylococcal strains resistant to 5--7 antibiotics differed from those resistant to a less number of the drugs by greater manifestation of the pathogenicity properties: lecithinase, hyaluronidase and hemolytic activity.     

Cytoplasmic membrane proteins of spectinomycin-susceptible and -resistant strains of Neisseria gonorrhoeae.

Cytoplasmic membranes were isolated and examined from two spectinomycin-susceptible and three spectinomycin-resistant clinical strains of Neisseria gonorrhoeae. A laboratory-derived spectinomycin-resistant mutant, obtained by serial passage on gradually increasing concentrations of the antibiotic, and a susceptible revertant, spontaneously arising from one of the resistant clinical strains, were also studied. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis revealed that a major protein, comprising about 7% of total cytoplasmic membrane protein (molecular weight 24,000), was absent in the three clinically isolated spectinomycin-resistant strains. In a revertant, this protein reappeared. During treatment of one of the susceptible strains with spectinomycin, the protein disappeared. However, this correlation was not maintained in the laboratory-derived spectinomycin-resistant mutant. This mutant was of comparable resistant to the clinical isolates, but the 24,000-molecular-weight protein was present in normal quantities. In addition, spectinomycin resistant in clinical isolates was variable compared with stable resistance exhibited by the laboratory-derived mutant. These findings suggested that differences in laboratory-derived versus clinical spectinomycin resistance may be due to different types of resistance mutations.     

Construction of a yeast mutant lacking the mitochondrial nuclease.

The nuclear gene from Saccharomyces cerevisiae that encodes the major mitochondrial nuclease was cloned. Gene sequences were identified from a lambda gt11 library by antibodies specific to the mitochondrial nuclease. DNA from the phage recombinant was used to isolate the entire nuclease gene from a plasmid library. Yeast strains containing the nuclease gene on a multicopy plasmid vector overproduced mitochondrial nuclease 20-40 times relative to a wild-type strain. Strains containing a null allele of the nuclease gene lacked all traces of mitochondrial nuclease. Both cell types, however, were phenotypically wild-type indicating that the nuclease is not an essential enzyme for mitochondrial function. The locus encoding the mitochondrial nuclease is termed NUC1.