scholarly journals Investigation of Macrolide Resistance Genotypes in Mycoplasma bovis Isolates from Canadian Feedlot Cattle

Pathogens ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 622 ◽  
Author(s):  
Andrea Kinnear ◽  
Tim A. McAllister ◽  
Rahat Zaheer ◽  
Matthew Waldner ◽  
Antonio C. Ruzzini ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Feedlot Cattle ◽  
23S Rrna ◽  
Dilution Method ◽  
Mycoplasma Bovis ◽  
Rrna Gene ◽  
Single Mutation ◽  
E Coli ◽  
23S Rrna Gene ◽  
Chronic Pneumonia

Mycoplasma bovis is associated with bovine respiratory disease (BRD) and chronic pneumonia and polyarthritis syndrome (CPPS) in feedlot cattle. No efficacious vaccines for M. bovis exist; hence, macrolides are commonly used to control mycoplasmosis. Whole genome sequences of 126 M. bovis isolates, derived from 96 feedlot cattle over 12 production years, were determined. Antimicrobial susceptibility testing (AST) of five macrolides (gamithromycin, tildipirosin, tilmicosin, tulathromycin, tylosin) was conducted using a microbroth dilution method. The AST phenotypes were compared to the genotypes generated for 23S rRNA and the L4 and L22 ribosomal proteins. Mutations in domains II (nucleotide 748; E. coli numbering) and V (nucleotide 2059 and 2060) of the 23S rRNA (rrl) gene alleles were associated with resistance. All isolates with a single mutation at Δ748 were susceptible to tulathromycin, but resistant to tilmicosin and tildipirosin. Isolates with mutations in both domain II and V (Δ748Δ2059 or Δ748Δ2060) were resistant to all five macrolides. However, >99% of isolates were resistant to tildipirosin and tilmicosin, regardless of the number and positions of the mutations. Isolates with a Δ748 mutation in the 23S rRNA gene and mutations in L4 and L22 were resistant to all macrolides except for tulathromycin.

scholarly journals Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

2021 ◽  
Author(s):  
J G E Laumen ◽  
S S Manoharan-Basil ◽  
E Verhoeven ◽  
S Abdellati ◽  
I De Baetselier ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Ribosomal Proteins ◽  
Efflux Pump ◽  
23S Rrna ◽  
Rrna Gene ◽  
23S Rrna Gene ◽  
Evolution Of Resistance ◽  
High Level ◽  
Azithromycin Resistance ◽  
Level Resistance

Abstract Background The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. Objectives To characterize the genetic pathways leading to high-level azithromycin resistance. Methods A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. Results Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. Conclusions This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

scholarly journals Macrolide Resistance in Campylobacter jejuni and Campylobacter coli: Molecular Mechanism and Stability of the Resistance Phenotype

2005 ◽  
Vol 49 (7) ◽  
pp. 2753-2759 ◽  
Author(s):  
Amera Gibreel ◽  
Veronica N. Kos ◽  
Monika Keelan ◽  
Cathy A. Trieber ◽  
Simon Levesque ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Target Gene ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Resistance Level ◽  
Resistance Phenotype ◽  
E Coli ◽  
23S Rrna Gene

ABSTRACT A collection of 23 macrolide-resistant Campylobacter isolates from different geographic areas was investigated to determine the mechanism and stability of macrolide resistance. The isolates were identified as Campylobacter jejuni or Campylobacter coli based on the results of the hippurate biochemical test in addition to five PCR-based genotypic methods. Three point mutations at two positions within the peptidyl transferase region in domain V of the 23S rRNA gene were identified. About 78% of the resistant isolates exhibited an A→G transition at Escherichia coli equivalent base 2059 of the 23S rRNA gene. The isolates possessing this mutation showed a wide range of erythromycin and clarithromycin MICs. Thus, this mutation may incur a greater probability of treatment failure in populations infected by resistant Campylobacter isolates. Another macrolide-associated mutation (A→C transversion), at E. coli equivalent base 2058, was detected in about 13% of the isolates. An A→G transition at a position cognate with E. coli 23S rRNA base 2058, which is homologous to the A2142G mutation commonly described in Helicobacter pylori, was also identified in one of the C. jejuni isolates examined. In the majority of C. jejuni isolates, the mutations in the 23S rRNA gene were homozygous except in two cases where the mutation was found in two of the three copies of the target gene. Natural transformation demonstrated the transfer of the macrolide resistance phenotype from a resistant Campylobacter isolate to a susceptible Campylobacter isolate. Growth rates of the resulting transformants containing A-2058→C or A-2059→G mutations were similar to that of the parental isolate. The erythromycin resistance of six of seven representative isolates was found to be stable after successive subculturing in the absence of erythromycin selection pressure regardless of the resistance level, the position of the mutation, or the number of the mutated copies of the target gene. One C. jejuni isolate showing an A-2058→G mutation, however, reverted to erythromycin and clarithromycin susceptibility after 55 subcultures on erythromycin-free medium. Investigation of ribosomal proteins L4 and L22 by sequence analysis in five representative isolates of C. jejuni and C. coli demonstrated no significant macrolide resistance-associated alterations in either the L4 or the L22 protein that might explain either macrolide resistance or enhancement of the resistance level.

Antimicrobial susceptibility and mechanisms of high-level macrolide resistance in clinical isolates of Moraxella nonliquefaciens

2014 ◽  
Vol 63 (2) ◽  
pp. 242-247 ◽  
Author(s):  
Shotaro Nonaka ◽  
Kosuke Matsuzaki ◽  
Tomoya Kazama ◽  
Hiroyuki Nishiyama ◽  
Yoko Ida ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Ribosomal Proteins ◽  
Macrolide Resistance ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Rrna Gene ◽  
Efflux System ◽  
Strong Activity ◽  
23S Rrna Gene ◽  
High Level

We investigated antimicrobial susceptibility and the molecular mechanism involved in conferring high-level macrolide resistance in 47 clinical isolates of Moraxella nonliquefaciens from Japan. Antimicrobial susceptibility was determined using Etest and agar dilution methods. Thirty-two erythromycin-non-susceptible strains were evaluated for the possibility of clonal spreading, using PFGE. To analyse the mechanism related to macrolide resistance, mutations in the 23S rRNA gene and the ribosomal proteins, and the presence of methylase genes were investigated by PCR and sequencing. The efflux system was examined using appropriate inhibitors. Penicillin, ampicillin, amoxicillin, cefixime, levofloxacin and antimicrobials containing β-lactamase inhibitors showed strong activity against 47 M. nonliquefaciens isolates. Thirty-two (68.1 %) of the 47 isolates showed high-level MICs to macrolides (MIC ≥128 mg l−1) and shared the A2058T mutation in the 23S rRNA gene. The geometric mean MIC to macrolides of A2058T-mutated strains was significantly higher than that of WT strains (P<0.0001). Thirty-two isolates with high-level macrolide MICs clustered into 30 patterns on the basis of the PFGE dendrogram, indicating that the macrolide-resistant strains were not clonal. In contrast, no common mutations of the ribosomal proteins or methylase genes, or overproduction of the efflux system were observed in A2058T-mutated strains. Moreover, of the 47 M. nonliquefaciens strains, 43 (91.5 %) were bro-1 and 4 (8.5 %) were bro-2 positive. Our results suggest that most M. nonliquefaciens clinical isolates show high-level macrolide resistance conferred by the A2058T mutation in the 23S rRNA gene. This study represents the first characterization of M. nonliquefaciens.

scholarly journals New Site of Modification of 23S rRNA Associated with Clarithromycin Resistance of Helicobacter pylori Clinical Isolates

2002 ◽  
Vol 46 (12) ◽  
pp. 3765-3769 ◽  
Author(s):  
Carla Fontana ◽  
Marco Favaro ◽  
Silvia Minelli ◽  
Anna Angela Criscuolo ◽  
Antonio Pietroiusti ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
23S Rrna ◽  
Dilution Method ◽  
Rrna Gene ◽  
Functional Sites ◽  
Biopsy Specimens ◽  
Clarithromycin Resistance ◽  
Pcr Product ◽  
23S Rrna Gene ◽  
H Pylori

ABSTRACT Resistance of Helicobacter pylori to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve H. pylori eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from H. pylori isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for H. pylori isolation. Eighty-six gastric biopsy specimens yielded H. pylori-positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 μg/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an HhaI restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates.

scholarly journals Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

2020 ◽  
Author(s):  
J.G.E. Laumen ◽  
S.S. Manoharan-Basil ◽  
E Verhoeven ◽  
S Abdellati ◽  
I De Baetselier ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Ribosomal Proteins ◽  
Efflux Pump ◽  
23S Rrna ◽  
Rrna Gene ◽  
23S Rrna Gene ◽  
Evolution Of Resistance ◽  
High Level ◽  
Azithromycin Resistance ◽  
Level Resistance

AbstractObjectivesThe prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. The aim of this study was to characterize the genetic pathways leading to high-level azithromycin resistance.MethodsA customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing.ResultsWithin 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low-to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE-encoded efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA - mainly the well-known A2059G and C2611T mutations, but also at position A2058G.ConclusionsThis study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

scholarly journals Association of Metronidazole Resistance and Natural Competence in Helicobacter pylori

2002 ◽  
Vol 46 (5) ◽  
pp. 1564-1567 ◽  
Author(s):  
Yu-Ching Yeh ◽  
Kai-Chin Chang ◽  
Jyh-Chin Yang ◽  
Chi-Tai Fang ◽  
Jin-Town Wang
Keyword(s):  
Helicobacter Pylori ◽  
Natural Transformation ◽  
23S Rrna ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Rrna Gene ◽  
Natural Competence ◽  
Metronidazole Resistance ◽  
23S Rrna Gene ◽  
Agar Dilution

ABSTRACT To study whether the capability of horizontal DNA transfer is associated with metronidazole resistance in Helicobacter pylori, a total of 81 clinical isolates were tested for MICs of metronidazole (MTZ). The MIC assays were performed by using the E-test and reconfirmed by the agar dilution method. Natural competence assays were performed by transferring a chloramphenicol acetyltransferase cassette and a 23S rRNA gene from a clarithromycin-resistant strain (with an A-to-G mutation at nucleotide 2143) by using natural transformation. Of the 81 isolates, 65 (80.2%) were naturally competent while 16 were not. Among the 65 naturally competent strains, 39 (60%) were highly resistant to MTZ (MICs, >32 μg/ml) while only 2 of 16 (12.5%) noncompetent strains were highly MTZ resistant (P, <0.001). Therefore, there is an association between natural competence and MTZ resistance.

scholarly journals Mutational and Transcriptomic Changes Involved in the Development of Macrolide Resistance in Campylobacter jejuni

2012 ◽  
Vol 57 (3) ◽  
pp. 1369-1378 ◽  
Author(s):  
Haihong Hao ◽  
Zonghui Yuan ◽  
Zhangqi Shen ◽  
Jing Han ◽  
Orhan Sahin ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Dna Microarray ◽  
Ribosomal Proteins ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
23S Rrna Gene ◽  
Resistant Mutants ◽  
Antibiotic Efflux

ABSTRACTMacrolide antibiotics are important for clinical treatment of infections caused byCampylobacter jejuni. Development of resistance to this class of antibiotics inCampylobacteris a complex process, and the dynamic molecular changes involved in this process remain poorly defined. Multiple lineages of macrolide-resistant mutants were selected by stepwise exposure ofC. jejunito escalating doses of erythromycin or tylosin. Mutations in target genes were determined by DNA sequencing, and the dynamic changes in the expression of antibiotic efflux transporters and the transcriptome ofC. jejuniwere examined by real-time reverse transcription-PCR, immunoblotting, and DNA microarray analysis. Multiple types of mutations in ribosomal proteins L4 and L22 occurred early during stepwise selection. On the contrary, the mutations in the 23S rRNA gene, mediating high resistance to macrolides, were observed only in the late-stage mutants. Upregulation of antibiotic efflux genes was observed in the intermediately resistant mutants, and the magnitude of upregulation declined with the occurrence of mutations in the 23S rRNA gene. DNA microarray analysis revealed the differential expression of 265 genes, most of which occurred in the intermediate mutant, including the upregulation of genes encoding ribosomal proteins and the downregulation of genes involved in energy metabolism and motility. These results indicate (i) that mutations in L4 and L22 along with temporal overexpression of antibiotic efflux genes precede and may facilitate the development of high-level macrolide resistance and (ii) that the development of macrolide resistance affects the pathways important for physiology and metabolism inC. jejuni, providing an explanation for the reduced fitness of macrolide-resistantCampylobacter.

scholarly journals Restriction Profiling of 23S Microheterogenic Ribosomal Repeats for Detection and Characterizing ofE. coliand Their Clonal, Pathogenic, and Phylogroups

2015 ◽  
Vol 2015 ◽  
pp. 1-11
Author(s):  
Parvathi Jayasree Rajagopalan Nair ◽  
Sunita Singh
Keyword(s):  
Cost Effective ◽  
23S Rrna ◽  
Rrna Gene ◽  
Type I ◽  
Microbial Identification ◽  
E Coli ◽  
Type Iv ◽  
Cost Effective Approach ◽  
23S Rrna Gene ◽  
K 12

Correlating ribosomal microheterogenicity with unique restriction profiles can prove to be an efficacious and cost-effective approach compared with sequencing for microbial identification. An attempt to peruse restriction profiling of 23S ribosomal assemblage was ventured; digestion patterns withBfaI discriminatedE. colifrom its colony morphovars, whileHaeIII profiles assisted in establishing distinct clonal groups. Among the gene pool of 399 ribosomal sequences extrapolated from 57E. coligenomes, varying degree of predominance (I > III > IV > II) ofHaeIII pattern was observed. This was also corroborated in samples collected from clinical, commensal, and environmental origin. K-12 and its descendants showed type I pattern whereasE. coli-B and its descendants exhibited type IV, both of these patterns being exclusively present inE. coli. A near-possible association between phylogroups andHaeIII profiles with presumable correlation between the clonal groups and different pathovars was established. The generic nature, conservation, and barcode gap of 23S rRNA gene make it an ideal choice and substitute to 16S rRNA gene, the most preferred region for molecular diagnostics in bacteria.

scholarly journals Intraspecific Diversity of the 23S rRNA Gene and the Spacer Region Downstream in Escherichia coli

1999 ◽  
Vol 181 (9) ◽  
pp. 2703-2709 ◽  
Author(s):  
Ana I. Antón ◽  
Antonio J. Martínez-Murcia ◽  
Francisco Rodríguez-Valera
Keyword(s):  
Escherichia Coli ◽  
Gene Families ◽  
Intraspecific Diversity ◽  
Rrna Genes ◽  
23S Rrna ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Enzyme Electrophoresis ◽  
Single Mutation ◽  
23S Rrna Gene

ABSTRACT The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection. The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the rrs gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected. The mode of variation of the rrl gene seems to be quite similar to that of therrs gene. Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation. The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3′ end.

scholarly journals Time-Kill Study of the Activity of Telithromycin against Macrolide-Resistant Streptococcus pneumoniae Isolates with 23S rRNA Mutations and Changes in Ribosomal Proteins L4 and L22

2005 ◽  
Vol 49 (7) ◽  
pp. 3011-3013 ◽  
Author(s):  
Ralf René Reinert ◽  
Adnan Al-Lahham
Keyword(s):  
Streptococcus Pneumoniae ◽  
Ribosomal Proteins ◽  
23S Rrna ◽  
Rrna Gene ◽  
23S Rrna Gene ◽  
Time Kill

ABSTRACT By use of a time-kill methodology, the antipneumococcal activity of telithromycin was determined against macrolide-resistant S. pneumoniae isolates having mutations in the 23S rRNA gene and changes in the ribosomal proteins L4 and L22. Telithromycin had MICs ranging between 0.03 and 0.25 μg/ml and was bactericidal against four of seven strains after 24 h at two times the MIC.

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