scholarly journals Analysis of methods commonly used for glycopeptide and oxazolidinone susceptibility testing in Enterococcus faecium isolates

2010 ◽  
Vol 59 (6) ◽  
pp. 672-678 ◽  
Author(s):  
Giammarco Raponi ◽  
Maria Cristina Ghezzi ◽  
Giovanni Gherardi ◽  
Giulia Lorino ◽  
Giordano Dicuonzo
Keyword(s):  
Enterococcus Faecium ◽  
Susceptibility Testing ◽  
23S Rrna ◽  
Rrna Gene ◽  
Disc Diffusion ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
Vitek 2 ◽  
Resistance Rates ◽  
The Phoenix

The susceptibility to teicoplanin, vancomycin and linezolid of 30 clinical isolates of Enterococcus faecium was tested by Vitek 2, Phoenix, Etest, broth microdilution and disc diffusion tests. The vanA and vanB resistance genes and the 23S rRNA gene G2576T mutation were detected by PCR and PCR-RFLP, respectively. Resistance rates to teicoplanin ranged from 3 % for Vitek 2 to 57.6 % for the Phoenix test, and those to vancomycin ranged from 56.7 % for Vitek 2 to 86.7 % for the Phoenix test. Only two out of 25 strains carrying the vanA gene were univocally recognized as the VanA phenotype. The only strain with the G2576T mutation did not carry the vanA gene and showed resistance to linezolid by the disc diffusion, Vitek 2 and broth dilution methods (MIC >8 μg ml−1), but was susceptible when tested with the Phoenix test and Etest (MIC ≤4 μg ml−1). Therefore, the resistance to glycopeptides and linezolid was not univocally detected by the susceptibility testing methods used in this study.

Co-infection with two different strains of Bordetella pertussis in an infant

2008 ◽  
Vol 57 (3) ◽  
pp. 388-391 ◽  
Author(s):  
Pamela K. Cassiday ◽  
Melissa Tobin-D'Angelo ◽  
J. Renee Watson ◽  
Kai-Hui Wu ◽  
Mahin M. Park ◽  
...  
Keyword(s):  
Bordetella Pertussis ◽  
Gene Sequence ◽  
Profile Analysis ◽  
23S Rrna ◽  
Rrna Gene ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
History Of ◽  
Simultaneous Infection ◽  
Different Strains

We report co-infection with two phenotypically and genotypically distinct strains of Bordetella pertussis in an infant male hospitalized with a 2-week history of cough, paroxysms and vomiting. Colonies from the two B. pertussis phenotypes were isolated and evaluated by PFGE profile analysis, gene sequence typing and PCR-RFLP of a portion of the 23S rRNA gene. These results demonstrated simultaneous infection with two different strains of B. pertussis.

scholarly journals Geographic Distribution and Genetic Diversity of Ceanothus-Infective FrankiaStrains

1999 ◽  
Vol 65 (4) ◽  
pp. 1378-1383 ◽  
Author(s):  
Nancy J. Ritchie ◽  
David D. Myrold
Keyword(s):  
Root Nodules ◽  
Intergenic Spacer ◽  
Taxonomic Diversity ◽  
Molecular Techniques ◽  
23S Rrna ◽  
Rrna Gene ◽  
Sample Collection ◽  
Pcr Product ◽  
Pcr Rflp ◽  
23S Rrna Gene

ABSTRACT Little is known about Ceanothus-infectiveFrankia strains because no Frankia strains that can reinfect the host plants have been isolated fromCeonothus spp. Therefore, we studied the diversity of theCeonothus-infective Frankia strains by using molecular techniques. Frankia strains inhabiting root nodules of nine Ceanothus species were characterized. The Ceanothus species used represent the taxonomic diversity and geographic range of the genus; therefore, the breadth of the diversity of Frankia strains that infectCeanothus spp. was studied. DNA was amplified directly from nodular material by using the PCR. The amplified region included the 3′ end of the 16S rRNA gene, the intergenic spacer, and a large portion of the 23S rRNA gene. A series of restriction enzyme digestions of the PCR product allowed us to identify PCR-restriction fragment length polymorphism (RFLP) groups among theCeanothus-infective Frankia strains tested. Twelve different enzymes were used, which resulted in four different PCR-RFLP groups. The groups did not follow the taxonomic lines of the Ceanothus host species. Instead, theFrankia strains present were related to the sample collection locales.

scholarly journals Genetic diversity and phylogeny of rhizobia isolated from agroforestry legume species in southern Ethiopia

2005 ◽  
Vol 55 (4) ◽  
pp. 1439-1452 ◽  
Author(s):  
Endalkachew Wolde-meskel ◽  
Zewdu Terefework ◽  
Åsa Frostegård ◽  
Kristina Lindström
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Genes ◽  
Partial Sequence ◽  
23S Rrna ◽  
Southern Ethiopia ◽  
Rrna Gene ◽  
Pcr Rflp ◽  
23S Rrna Gene

The genetic diversity within 195 rhizobial strains isolated from root nodules of 18 agroforestry species (15 woody and three herbaceous legumes) growing in diverse ecoclimatic zones in southern Ethiopia was investigated by using PCR–RFLP of the ribosomal operon [16S rRNA gene, 23S rRNA gene and the internal transcribed spacer (ITS) region between the 16S rRNA and 23S rRNA genes] and 16S rRNA gene partial sequence (800 and 1350 bp) analyses. All of the isolates and the 28 reference strains could be differentiated by using these methods. The size of the ITS varied among test strains (500–1300 bp), and 58 strains contained double copies. UPGMA dendrograms generated from cluster analyses of the 16S and 23S rRNA gene PCR–RFLP data were in good agreement, and the combined distance matrices delineated 87 genotypes, indicating considerable genetic diversity among the isolates. Furthermore, partial sequence analysis of 67 representative strains revealed 46 16S rRNA gene sequence types, among which 12 were 100 % similar to those of previously described species and 34 were novel sequences with 94–99 % similarity to those of recognized species. The phylogenetic analyses suggested that strains indigenous to Ethiopia belonged to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Methylobacterium, Rhizobium and Sinorhizobium. Many of the rhizobia isolated from previously uninvestigated indigenous woody legumes had novel 16S rRNA gene sequences and were phylogenetically diverse. This study clearly shows that the characterization of symbionts of unexplored legumes growing in previously unexplored biogeographical areas will reveal additional diversity.

scholarly journals Rapid Identification of Listeria Species by Using Restriction Fragment Length Polymorphism of PCR-Amplified 23S rRNA Gene Fragments

2003 ◽  
Vol 69 (11) ◽  
pp. 6386-6392 ◽  
Author(s):  
Delphine Paillard ◽  
Véronique Dubois ◽  
Robert Duran ◽  
Fany Nathier ◽  
Catherine Guittet ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
23S Rrna ◽  
Rrna Gene ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
Restriction Fragment Length

ABSTRACT A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.

scholarly journals PCR-RFLP detection of point mutations A2143G and A2142G in 23S rRNA gene conferring resistance to clarithromycin in Helicobacter pylori strains.

2014 ◽  
Vol 61 (2) ◽  
Author(s):  
Karolina Klesiewicz ◽  
Paweł Nowak ◽  
Elżbieta Karczewska ◽  
Iwona Skiba ◽  
Izabela Wojtas-Bonior ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Point Mutations ◽  
Rflp Analysis ◽  
23S Rrna ◽  
Rrna Gene ◽  
Efficient Management ◽  
Clarithromycin Resistance ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
H Pylori

The occurrence of clarithromycin resistance among Helicobacter pylori strains is a major cause of the treatment failure. Resistance to this drug is conferred by point mutations in 23S rRNA gene and the most prevalent mutations are A2143G and A2142G. The aim of the study was to evaluate the occurrence of A2143G and A2142G mutations in a group of H. pylori strains resistant to clarithromycin. The study included 21 clarithromycin-resistant H. pylori strains collected between 2006 and 2009 in southern Poland. Resistance to clarithromycin was quantitatively tested with the E-test to determine the minimal inhibitory concentration (MIC value). The point mutations of H. pylori isolates were detected by PCR followed by RFLP analysis. The MIC values for clarithromycin for the analyzed strains ranged from 1.5 mg/L to 64 mg/L. Nine H. pylori strains exhibited A2143G mutation and A2142G mutation was found in 9 isolates as well. The results of RFLP analysis of 3 clarithromycin-resistant strains were negative for both mutations. The average MIC values for A2143G and A2142G mutants were 6 and 30 mg/L, respectively. Frequencies of A2143G and A2142G mutations were the same in all isolates tested. Strains with A2143G mutation exhibited lower MIC values than A2142G mutants. Application of PCR-RFLP method for detection of clarithromycin resistance allows for better and more efficient management of H. pylori infections.

scholarly journals Impact of Clarithromycin Resistance on Eradication of Helicobacter pylori in Infected Adults

2005 ◽  
Vol 49 (4) ◽  
pp. 1600-1603 ◽  
Author(s):  
Jong Hwa Lee ◽  
Ji-Hyun Shin ◽  
Im Hwan Roe ◽  
Seung Ghyu Sohn ◽  
Jung Hun Lee ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Drug Therapy ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Gastric Biopsy ◽  
Antimicrobial Susceptibility Testing ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
23S Rrna Gene

ABSTRACT The outcome of Helicobacter pylori infection was analyzed in 114 dyspeptic patients treated with triple-drug therapy including clarithromycin. Clarithromycin resistance (in 20.2% of our isolates) was mainly caused by an A2142G mutation in the 23S rRNA gene of H. pylori. H. pylori eradication was obtained in all patients with clarithromycin-susceptible isolates but not in any patients with clarithromycin-resistant isolates (P = 0.0001). Therefore, it would be useful to conduct H. pylori antimicrobial susceptibility testing of the first gastric biopsy culture before choosing the first three drugs for therapy of infected patients.

scholarly journals Clinical-Use-Associated Decrease in Susceptibility of Vancomycin-Resistant Enterococcus faecium to Linezolid: a Comparison with Quinupristin-Dalfopristin

2004 ◽  
Vol 48 (9) ◽  
pp. 3583-3585 ◽  
Author(s):  
Issam I. Raad ◽  
Hend A. Hanna ◽  
Ray Y. Hachem ◽  
Tanya Dvorak ◽  
Rebecca B. Arbuckle ◽  
...  
Keyword(s):  
Gene Mutation ◽  
Enterococcus Faecium ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clinical Use ◽  
Vancomycin Resistant Enterococcus ◽  
23S Rrna Gene ◽  
Vancomycin Resistant ◽  
Domain V

ABSTRACT The susceptibility of 135 vancomycin-resistant Enterococcus faecium bacteremic isolates to linezolid and quinupristin-dalfopristin was determined. All were susceptible to linezolid, while 88% were susceptible to quinupristin-dalfopristin prior to the clinical use of the drugs at our hospital. More than 6 months after their clinical use, a decrease in susceptibility was noted for only linezolid at 83%. This was related in part to a single G2576U gene mutation in domain V of the 23S rRNA gene.

scholarly journals Antimicrobial susceptibility and clarithromycin resistance patterns of Helicobacter pylori clinical isolates in Vietnam

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 671 ◽  
Author(s):  
Camelia Quek ◽  
Son T. Pham ◽  
Kieu T. Tran ◽  
Binh T. Pham ◽  
Loc V. Huynh ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antimicrobial Resistance ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
Resistance Patterns ◽  
23S Rrna Gene ◽  
H Pylori

Helicobacter pylori is a gastric pathogen that causes several gastroduodenal disorders such as peptic ulcer disease and gastric cancer.  Eradication efforts of H. pylori are often hampered by antimicrobial resistance in many countries, including Vietnam.  Here, the study aimed to investigate the occurrence of antimicrobial resistance among H. pylori clinical isolates across 13 hospitals in Vietnam.  The study further evaluated the clarithromycin resistance patterns of H. pylori strains.  In order to address the study interests, antimicrobial susceptibility testing, epsilometer test and PCR-based sequencing were performed on a total of 193 strains isolated from patients, including 136 children (3–15 years of age) and 57 adults (19–69 years of age).  Antimicrobial susceptibility testing showed that the overall resistance to amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline was 10.4%, 85.5%, 24.4%, 37.8%, and 23.8% respectively.  The distribution of minimum inhibitory concentrations (MICs) of clarithromycin-resistant strains was 85.5% with MIC >0.5 μg/mL.  The majority of the clarithromycin resistant isolates (135 of 165 subjects) have MICs ranging from 2 μg/mL to 16 μg/mL.  Furthermore, sequencing detection of mutations in 23S rRNA gene revealed that strains resistant and susceptible to clarithromycin contained both A2143G and T2182C mutations.  Of all isolates, eight clarithromycin-resistant isolates (MIC >0.5 μg/mL) had no mutations in the 23S rRNA gene.  Collectively, these results demonstrated that a proportion of clarithromycin-resistant H. pylori strains, which are not related to the 23S rRNA gene mutations, could be potentially related to other mechanisms such as the presence of an efflux pump or polymorphisms in the CYP2C19 gene.  Therefore, the present study suggests that providing susceptibility testing prior to treatment or alternative screening strategies for antimicrobial resistance is important for future clinical practice.  Further studies on clinical guidelines and treatment efficacy are pivotal for successful eradication of H. pylori infection.

STUDY ON POINT MUTATIONS AT POSITIONS 2132 AND 2143 IN 23S RRNA GENE OF HELICOBACTER PYLORI AMONG PATIENTS WITH CHRONIC GASTRITIS

2016 ◽  
pp. 12-20
Author(s):  
Thi Minh Thi Ha ◽  
Van Huy Tran ◽  
Viet Nhan Nguyen ◽  
Thanh Hoa Nguyen ◽  
Phan Tuong Quynh Le
Keyword(s):  
Helicobacter Pylori ◽  
Chronic Gastritis ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
History Of ◽  
H Pylori

Background: Clarithromycin resistance in Helicobacter pylori has been found to be associated with point mutations at positions 2142 and 2143 in 23SrRNA gene. The aims of this study were: (1) to determine the rates of point mutations A2143G, A2142G and A2142C in 23SrRNA gene of H. pylori among patients with chronic gastritis by PCR-RFLP technique; and (2) to assessthe association between these mutations and some clinical, endoscopic and histopathological characteristics of chronic gastritis. Patients and methods: two hundreds and twenty six patients with H. pylori-positive chronic gastritis were determined A2143G, A2142G and A2142C mutations by PCR-RFLP technique with DNA extracted from endoscopic biopsy specimens of gastric mucosa. Results: The rate of point mutations at positions 2142 and 2143 in 23S rRNA gene of H. pylori was 35.4% in total, the A2143G and A2142G mutationsaccounted for 92.5% and 7.5% of all point mutations, respectively. No A2142C mutation was found. These mutations were not associated with age, gender,distribution of gastritis, and the presence of atrophic gastritis. The rate of A2143G mutation in groups with and without a history of clarithromycin treatment were 44.9% and 24.8%, respectively (p = 0,0065). The A2142G mutation was associated with intestinal metaplasia and/or dysplasia. Conclusion: The point mutations at positions 2142 and 2143 in 23S rRNA gene were found at a high rate in H. pylori strains amongpatients with chronic gastritis, with the absolute predominance of A2143G mutation. The A2143G mutation was associated with a history of clarithromycin treatment. Key words: 23S rRNA gene, Helicobacter pylori, A2143G, A2142G, A2142C mutation, clarithromycin resistance, chronic gastritis.

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