Hydrogel Droplet Microarray for Genotyping Antimicrobial Resistance Determinants in Neisseria gonorrhoeae Isolates

A multiplex assay based on a low-density hydrogel microarray was developed to identify genomic substitutions in N. gonorrhoeae that determine resistance to the currently recommended treatment agents ceftriaxone and azithromycin and the previously used drugs penicillin, tetracycline, and ciprofloxacin. The microarray identifies 74 drug resistance determinants in the N. gonorrhoeae penA, ponA, porB, gyrA, parC, rpsJ, mtrR, blaTEM, tetM, and 23S rRNA genes. The hydrogel elements were formed by automated dispensing of nanoliter-volume droplets followed by UV-induced copolymerization of NH2-containing oligonucleotides with gel-forming monomers. Polybutylene terephthalate plates without special modifications were used as microarray substrates. Sequences and concentrations of immobilized oligonucleotides, gel composition, and hybridization conditions were carefully selected, and the median discrimination ratio ranged from 2.8 to 29.4, allowing unambiguous identification of single-nucleotide substitutions. The mutation identification results in a control sample of 180 N. gonorrhoeae isolates were completely consistent with the Sanger sequencing results. In total, 648 clinical N. gonorrhoeae isolates obtained in Russia during the last 5 years were analyzed and genotyped using these microarrays. The results allowed us to draw conclusions about the present situation with antimicrobial susceptibility of N. gonorrhoeae in Russia and demonstrated the possibility of using hydrogel microarrays to control the spread of antibiotic resistance.

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Distinct Types of rRNA Operons Exist in the Genome of the Actinomycete Thermomonospora chromogena and Evidence for Horizontal Transfer of an Entire rRNA Operon

Journal of Bacteriology ◽

10.1128/jb.181.17.5201-5209.1999 ◽

1999 ◽

Vol 181 (17) ◽

pp. 5201-5209 ◽

Cited By ~ 190

Author(s):

Wai Ho Yap ◽

Zhenshui Zhang ◽

Yue Wang

Keyword(s):

Evolutionary Relationship ◽

Comparative Sequence Analysis ◽

Rrna Genes ◽

23S Rrna ◽

Nucleotide Substitutions ◽

Thermophilic Actinomycete ◽

Related Organism ◽

Sequence Variations ◽

Nonrandom Distribution ◽

23S Rrna Genes

ABSTRACT We describe here the presence of two distinct types of rRNA operons in the genome of a thermophilic actinomycete Thermomonospora chromogena. The genome of T. chromogena contains six rRNA operons (rrn), of which four complete and two incomplete ones were cloned and sequenced. Comparative analysis revealed that the operon rrnB exhibits high levels of sequence variations to the other five nearly identical ones throughout the entire length of the operon. The coding sequences for the 16S and 23S rRNA genes differ by approximately 6 and 10%, respectively, between the two types of operons. Normal functionality ofrrnB is concluded on the basis of the nonrandom distribution of nucleotide substitutions, the presence of compensating nucleotide covariations, the preservation of secondary and tertiary rRNA structures, and the detection of correctly processed rRNAs in the cell. Comparative sequence analysis also revealed a close evolutionary relationship between rrnB operon of T. chromogena and rrnA operon of another thermophilic actinomycete Thermobispora bispora. We propose thatT. chromogena acquired rrnB operon fromT. bispora or a related organism via horizontal gene transfer.

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Demonstration of the absence of intervening sequences within 23S rRNA genes fromCampylobacter lari

Journal of Basic Microbiology ◽

10.1002/jobm.200800232 ◽

2009 ◽

Vol 49 (4) ◽

pp. 386-394 ◽

Cited By ~ 1

Author(s):

Akihiro Tazumi ◽

Yuki Kakinuma ◽

John E. Moore ◽

Cherie B. Millar ◽

Ikue Taneike ◽

...

Keyword(s):

Rrna Genes ◽

23S Rrna ◽

Intervening Sequences ◽

23S Rrna Genes

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tRNA genes are found between the 16S and 23S rRNA genes in Bacillus subtilis

Nucleic Acids Research ◽

10.1093/nar/10.5.1607 ◽

1982 ◽

Vol 10 (5) ◽

pp. 1607-1624 ◽

Cited By ~ 76

Author(s):

Kate Loughney ◽

Elsebet Lund ◽

James E. Dahlberg

Keyword(s):

Bacillus Subtilis ◽

Rrna Genes ◽

23S Rrna ◽

Trna Genes ◽

23S Rrna Genes

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Spontaneous Erythromycin Resistance Mutation in a 23S rRNA Gene, rrlA, of the Extreme Thermophile Thermus thermophilus IB-21

Journal of Bacteriology ◽

10.1128/jb.183.14.4382-4385.2001 ◽

2001 ◽

Vol 183 (14) ◽

pp. 4382-4385 ◽

Cited By ~ 9

Author(s):

Steven T. Gregory ◽

Jamie H. D. Cate ◽

Albert E. Dahlberg

Keyword(s):

Genetic Manipulation ◽

Thermus Thermophilus ◽

Resistance Mutation ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Erythromycin Resistance ◽

23S Rrna Gene ◽

Resistant Mutants ◽

23S Rrna Genes

ABSTRACT Spontaneous, erythromycin-resistant mutants of Thermus thermophilus IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons. The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism. This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes ofThermus.

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Heterogeneous Macrolide Resistance and Gene Conversion in the Pneumococcus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.1.359-361.2006 ◽

2006 ◽

Vol 50 (1) ◽

pp. 359-361 ◽

Cited By ~ 11

Author(s):

Nicole Wolter ◽

Anthony M. Smith ◽

David J. Farrell ◽

Keith P. Klugman

Keyword(s):

Streptococcus Pneumoniae ◽

Gene Conversion ◽

Clinical Isolate ◽

Resistant Strain ◽

Macrolide Resistance ◽

Fitness Cost ◽

Rrna Genes ◽

23S Rrna ◽

Resistant Phenotype ◽

23S Rrna Genes

ABSTRACT A macrolide-resistant clinical isolate of Streptococcus pneumoniae with 23S rRNA mutations showed a heterogeneous phenotype and genotype. The mutant 23S rRNA genes from this isolate transformed susceptible strain R6 to resistance. Culture of resistant strain R6 in the absence of antibiotic pressure showed gene conversion to occur between the four 23S rRNA alleles, resulting in reversion to susceptibility with the resistant phenotype showing a fitness cost. These data explain the disappearance on subculture of heterogeneous macrolide resistance in the pneumococcus.

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Analysis of 23S rRNA genes in metagenomes – A case study from the Global Ocean Sampling Expedition

Systematic and Applied Microbiology ◽

10.1016/j.syapm.2011.04.005 ◽

2011 ◽

Vol 34 (6) ◽

pp. 462-469 ◽

Cited By ~ 11

Author(s):

Pelin Yilmaz ◽

Renzo Kottmann ◽

Elmar Pruesse ◽

Christian Quast ◽

Frank Oliver Glöckner

Keyword(s):

Global Ocean ◽

Rrna Genes ◽

23S Rrna ◽

Global Ocean Sampling ◽

Ocean Sampling ◽

23S Rrna Genes

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Pathophysiology of Antibiotic Resistance: Clarithromycin

Canadian Journal of Gastroenterology ◽

10.1155/2000/140806 ◽

2000 ◽

Vol 14 (10) ◽

pp. 891-894 ◽

Cited By ~ 10

Author(s):

Diane E Taylor

Keyword(s):

Inhibitory Concentration ◽

Natural Transformation ◽

Rrna Genes ◽

23S Rrna ◽

Cross Resistance ◽

In Vitro Mutagenesis ◽

Clarithromycin Resistance ◽

23S Rrna Genes ◽

H Pylori

Resistance ofHelicobacter pylorito antibiotics ranges from 3% to 10% and may exceed these levels in some countries. The pathophysiology of clarithromycin resistance is reviewed, including the mode of action by which the antibiotic inhibits protein synthesis and the mechanism of resistance, which involves a mutation at position 2142 or 2143 in the V loop domain of the 23S rRNA genes. Mutations of A2142G confer a higher minimum inhibitory concentration than mutations of A2143G. The former demonstrate cross-resistance to macrolide, lincosamide and streptogramin antibiotics, whereas the latter are susceptible to streptogramin B. In vitro mutagenesis combined with natural transformation were used to create several types of clarithromycin-resistant mutants.H pyloristrains with A2142G and A2143G mutations had a higher growth rate than those with A2142C, A2143 or A2142T mutations. Data from this study indicate why clarithromycin-resistant clinical isolates ofH pyloriare more likely to have A2142G or A2143G mutations and only occasionally A2142C mutations.

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