Allele‑specific duplex polymerase chain reaction to differentiate Mycobacterium abscessus subspecies and to detect highly clarithromycin‑resistant isolates.

On the basis of the structural differences of erm, we used a duplex polymerase chain reaction (PCR) to differentiate Mycobacterium abscessus subsp. abscessus and subsp. massiliense isolates and to detect the point mutations of 23S rRNA gene that confer a high level of resistance to clarithromycin. Subsp. massiliense strains occupying almost half of the clinical isolates can be simply identified, and their clarithromycin susceptibility can be rapidly determined.

Linezolid resistance in methicillin-resistant Staphylococcus epidermidis strains isolated in Hangzhou ;area / 中华微生物学和免疫学杂志

Objective To investigate the mechanism of linezolid resistance in methicillin-resistant Staphylococcus epidermidis ( MRSE) strains isolated in Hangzhou area. Methods Twenty-three linezolid-re-sistant Staphylococcus epidermidis ( LRSE) strains were isolated from the patients with septicaemia, urinary tract infection or infectious pleuritis in several hospitals in Hangzhou area during May, 2013 to April, 2015. The minimal inhibition concentrations ( MICs) of thirteen different antibiotics against the LRSE strains were detected by using E-test. Pulsed-field gel electrophoresis ( PFGE) and cluster analysis were performed for homology analysis. Correlations between linezolid resistance in the LRSE strains and G2576T mutation at theⅤ functional region of 23S rRNA gene or cfr gene were determined by PCR and sequencing analysis. Re-sults The MICs of linezolid to 15 LRSE strains were higher than 256 μg/ml while to the other 8 LRSE strains were 6 or 8 μg/ml. All of the 23 LRSE strains were resistant to both oxacillin and cefoxitin. Among the LRSE strains with high linezolid MICs (MIC>256 μg/ml), LRSE1 and LRSE2, LRSE3-LRSE6 and LRSE9-LRSE12 respectively belonged to the same clone line and came from the same hospital. All of the 23 LRSE strains carried the cfr gene. Moreover, the G2576T mutation at theⅤfunctional region of 23S rRNA gene was detected in the 15 LRSE strains with high linezolid MICs ( MIC>256 μg/ml ) , but not in the 8 strains with lower linezolid MICs ( MIC=6 or 8 μg/ml) . Conclusion There are significant differences in linezolid resistance among LRSE strains isolated in Hangzhou area. The LRSE strains are methicillin-resist-ant coagulase negative Staphylococcus ( MRCNS) strains and widely distributed. The linezolid resistance in LRSE strains is related to the G2576T mutation at theⅤfunctional region of 23S rRNA gene and cfr gene.

Determinación de mutaciones de un solo nucleótido en el gen 23S rRNA de Helicobacter pylori relacionadas con resistencia a claritromicina en una población del departamento del Cauca, Colombia / Determination of single nucleotide mutations in the 23S rRNA gene of Helicobacter pylori related to clarithromycin resistance in a population from Cauca, Colombia

Introducción. La terapia antibiótica combinada para la erradicación de Helicobacter pylori debería basarse en los patrones locales de resistencia. Objetivo. Determinar la resistencia de H. pylori a claritromicina en una población del departamento del Cauca mediante la identificación de mutaciones en el gen 23S r RNA en ADN obtenido de biopsias gástricas. Materiales y métodos. Se incluyeron en el estudio 162 pacientes con dispepsia funcional. El gen 23S rRNA se amplificó por PCR y el patrón de mutaciones se identificó por secuenciación directa. Resultados. La frecuencia de resistencia a claritromicina fue de 4 %. La mutación A2143G del gen se encontró en cuatro pacientes (2,46 %) y la mutación A2142G, en tres pacientes (1,85 %). Conclusiones. El estudio encontró que el genotipo más frecuente en los especímenes positivos para H. pylori fue 2143G, seguido por A2142G. La prevalencia observada de resistencia de H. pylori fue baja; por lo tanto, se considera que el tratamiento con claritromicina es una opción válida para la erradicación de H. pylori en la población objeto de estudio.

Introduction: Antibiotic combination therapy for the eradication of Helicobacter pylori should be based on local resistance patterns. Objective: To d etermine the resistance of H. pylori to clarithromycin in a population from Cauca province, through the identification of mutations in the 23S rRNA gene in DNA from gastric biopsies. Materials and methods: A total of 162 patients with functional dyspepsia were included in the study. The 23S rRNA gene and the DNA from 162 gastric specimens were amplified by PCR, and the mutation pattern was identified by direct sequencing. Results: The frequency of clarithromycin resistance was 4%. A2143G mutation was found in four patients (2.46%) and A2142G mutation was found in three patients (1.85%). Conclusions: Our study shows that the most frequent genotype in H. pylori -positive specimens was A2143G, followed by A2142G. The observed resistance prevalence of H. pylori was low; thus, we consider that clarithromycin treatment is a valid option for H. pylori eradication in the study population.

The genomic identification of Colombian Acinetobacter baumannii clinical isolates by RFLP-PCR analysis of the 16S-23S rRNA gene spacer region / Identificación genómica de aislamientos colombianos de Acinetobacter baumannii mediante RFLP-PCR de la región intergénica espaciadora de los genes 16S y 23S rRNA

The 16S-23S rRNA gene intergenic spacer (ITS) was analysed by RFLP in this study to identify A. baumannii from 139 isolates from four hospitals (identified as A, B, C and D). One hundred and twenty of these isolates (86.3%) belonged to the A. baumannii species; those identified as being A. baumannii were found to be polyclonal (19 clone groups) when determining the genetic relationships, 16 of them being found in hospital C. Hospitals A, B and D shared two clone groups isolated during different years. This study describes a rapid and easy method for genospecies identification of Acinetobacter baumannii.

Con el objeto de identificar la genomoespecie Acinetobacter baumannii, se estudiaron 189 aislamientos pertenecientes al Complejo Acinetobacter baumannii-Acinetobacter calcoaceticus provenientes de cuatro hospitales colombianos (denominados A,B,C,D) mediante el análisis por RFLP-PCR de la región intergénica espaciador (ITS) de los genes 16S y 23S rRNA. Se encontraron 120 aislamientos (86.3%) pertenecientes a la especie A. baumannii. La estructura de la población fue policlonal, con 19 grupos clonales, 16 de los cuales se hallaron en el hospital C. En los hospitales A,B y D se encontraron 2 grupos clonales aislados durante diferentes años. En este estudio se propone un método rápido y fácil para la identificación de Acinetobacter baumannii.

Prevalence and Antimicrobial Susceptibility of Campylobacter coli Isolates from Swine

Swine is a common source of Campylobacter coli human gastroenteritis, for the treatment of which erythromycin and fluoroquinolones are recommended. The prevalence of antimicrobial-resistant C. coli differs significantly depending on countries. We investigated the prevalence of C. coli in swine from a farm in Buan-gun, Korea in 2010, and determined antimicrobial susceptibility of the isolates. Rectal swab specimens were used to inoculate Campylobacter Preston media and incubated microaerophilically at 42degrees C for 48 h. The species were identified by phenotypic tests and by detecting hipO and glyA genes. PCR was used to detect mutations of A2074C in 23S rRNA gene, and quinolone resistance-determining region (QRDR) of gyrA, which are associated with high level resistance to erythromycin, and with ciprofloxacin, respectively. Antimicrobial susceptibility was determined by the disk diffusion and agar dilution tests. Of the 100 specimens, 55 (55%) yielded C. coli, and 23 of them (41.8%) had A2074G mutation. A2074G mutated isolates showed the lowest MIC90 of imipenem, while those of ampicillin and clindamycin were relatively low. The majority of both A2074G mutation-positive and -negative isolate were susceptible to ampicillin, cefotaxime, and chloramphenicol. All isolates were resistant to ciprofloxacin, and had mutation in QRDR of gyrA. In conclusion, C. coli was detected in 55% of swine, and A2074G mutation was detected in 41.8% of the isolates. All isolates had gyrA mutation-mediated ciprofloxacin resistance.

Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6 percent presented nucleotide substitutions: A2142G (3.3 percent), T2182C (12.9 percent), G2224A (6.45 percent), T2215C (61.3 percent), A2192G (3.3 percent), G2204C (6.4 percent) and T2221C (6.4 percent). Of the mutations identified, four are known mutations related to cases of resistance and 16.1 percent are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

Mutations in the 23S rRNA Gene of Helicobacter pylori Associated with Clarithromycin Resistance

Among 12 clarithromycin-resistant Helicobacter pylori strains isolated in Guri, Korea, 8 showed an adenine to guanine mutation at position 2143 (formerly A2144G or E. coli 2059) in the 23S rRNA gene by the PCR-restriction fragment length polymorphism (RFLP) method. The remaining 4 strains, digested by neither BsaI nor BbsI, showed a thymine to cytosine mutation at position 2182 (T2182C) by direct sequencing of the PCR products. The T2182C mutants showed a tendency of higher levels of minimum inhibitory concentration to clarithromycin than the A2143G mutants. In conclusion, either the A2143G or the T2182C mutation was present in 100% of clarithromycin-resistant H. pylori isolates examined. The PCR-RFLP technique with restriction enzymes BbsI and BsaI was a rapid and relatively simple method to detect the clarithromycin resistance. But undigested isolates were quite frequent among our isolates (33.3%), the PCR-RFLP method with restriction enzymes BbsI and BsaI should not be used alone, and development of other rapid detection method for clarithromycin resistance is mandatory.