Implementación de un método molecular para la identificación de Mycoplasma pneumoniae resistente a macrólidos / Implementation of a molecular method for the detection of macrolide-resistant Mycoplasma pneumoniae

Introducción: Mycoplasma pneumoniae es causa frecuente de infecciones respiratorias en niños y jóvenes. Los macrólidos son la primera línea de tratamiento. La rápida emergencia de resistencia a estos antimicrobianos ha motivado el desarrollo de métodos moleculares para su detección en muestras clínicas positivas a este patógeno. Objetivo: Implementar un método de reacción en cadena de la polimerasa en tiempo real (RT-PCR) para la detección de resistencia a macrólidos a partir de muestras clínicas positivas a M. pneumoniae. Métodos: Se implementó una RT-PCR para la detección de las mutaciones A2058G y A2059G en el ARNr 23S de M. pneumoniae. Se analizaron 24 muestras clínicas positivas a M. pneumoniae, que provenían de pacientes con síntomas respiratorios. Se evaluó la sensibilidad, especificidad, repetibilidad y reproducibilidad de la RT-PCR. Resultados: La RT-PCR mostró un 100 % de especificidad para M. pneumoniae y un 92 % de sensibilidad, con un límite de detección de 2 copias/µL, que equivale a 10 copias/reacción. Además, se demostró la reproducibilidad y repetibilidad de estos resultados. Se obtuvo una correcta identificación de los genotipos salvaje y mutante, correspondientes a cada control. De las muestras clínicas positivas a M. pneumoniae, el 77,3 % (17/22) se identificó como sensible a macrólidos y el 22,7 % (5/22) como resistente. Conclusiones: La alta sensibilidad y especificidad del método de RT-PCR implementado permite que el Laboratorio Nacional de Referencia de Micoplasmas del Instituto de Medicina Tropical Pedro Kourí cuente con un método eficaz para el diagnóstico de M. pneumoniae resistente a macrólidos.

Introduction: Mycoplasma pneumoniae is a common cause of respiratory track infections in children and young adults. Macrolides are the first-line treatment. The rapid emergence of resistance to these antimicrobials has motivated the development of molecular methods for their detection in clinical samples positive for this pathogen. Objective: To implement a real-time polymerase chain reaction (RT-PCR) method for the detection of macrolide resistance in M. pneumoniae positive clinical samples. Methods: An RT-PCR was implemented to detect mutations A2058G and A2059G in 23S rRNA of M. pneumoniae. M. pneumoniae positive clinical samples from 24 patients with respiratory symptoms were analyzed. Sensitivity, specificity, repeatability and reproducibility of the RT-PCR assays were evaluated. Results: The RT-PCR assays showed 100% specificity to M. pneumoniae, and 92% sensitivity with a detection limit of 2 copies/µL, equivalent to 10 copies/reaction. Moreover, the repeatability and reproducibility of these results were demonstrated. Wild and mutant genotypes associated to each control were properly identified. Of the clinical samples positive for M. pneumoniae, 77.3% (17/22) were macrolide-sensitive and 22.7% (5/22) were macrolide-resistant. Conclusions: The high sensitivity and specificity of the RT-PCR method implemented provides the National Reference Laboratory of Mycoplasmas of the Institute of Tropical Medicine Pedro Kourí with an effective method for the diagnosis of macrolide-resistant M. pneumoniae.

Drug resistance and clarithromycin resistance gene mutations in Helicobacter pylori strains circulating in Changchun area / 中华微生物学和免疫学杂志

Objective To investigate the resistance of Helicobacter pylori (H. pylori) strains to common antibiotics and to analyze the sites of genetic mutations carried by clarithromycin-resistant strains in order to provide reference for selecting sensitive antibiotics against H. pylori and for providing individualized treatment for patients in Changchun area. Methods Drug resistance of H. pylori clinical isolates to common antibiotics was detected by disk dilution method. The 23S rRNA genes of clarithromycin-resistant strains were amplified by PCR and then sequenced to analyze the presence of mutations. Results In this study, 69 strains of H. pylori were successfully isolated with a positive rate of 23. 1％ . Results of the drug susceptibility test to seven commonly used antibiotics showed that there were 52. 2％ of the isolates resistant to clarithromy-cin, 47. 8％ to tinidazole, 37. 7％ to levofloxacin, 33. 3％ to tetracycline hydrochloride, 30. 4％ to furazoli-done, 30. 4％ to metronidazole and 5. 8％ to amoxicillin. Amoxicillin could continue to be used as a first-line antimicrobial agent. Seven mutation sites were found in the 23S rRNA genes carried by the clarithromy-cin-resistant strains, which were A1821G, G1826A, T1830C, G1940A, A2143G, T2182C and A2223G. The A2143G site mutation accounted for 54. 2％ and was the predominant mutation resulting in the resistance to clarithromycin of H. pylori strains circulating in this area. Conclusions The H. pylori strains isolated from patients with gastroduodenal diseases in Changchun area had a high resistance rate to clarithromycin, which was mainly caused by the A2143G mutation in 23S rRNA gene.

Relationship between macrolide resistance mutations in Mycoplasma pneumoniae and its genotype / 中华微生物学和免疫学杂志

Objective To analyze the relationship between macrolide resistance mutations in My-coplasma pneumoniae (Mp) and its genotype by multiple-locus variable-number tandem-repeat analysis (MLVA). Methods One hundred and forty-three Mp-positive specimens were collected in Beijing(54 col-lected at the Affiliated Children′s Hospital of the Capital Institute of Pediatrics),the United States(59 col-lected at four different geographical locations:Kansas City,Missouri;Seattle,Washington;New York,New York;Chicago,Illinois) and Australia(30 provided by the diagnostic laboratory at the Centre for Infectious Diseases and Microbiology Laboratory Services,Institute of Clinical Pathology and Medical Research,West-mead Hospital,Sydney). Nested PCR was used to detect mutations in 23S rRNA. A capillary electrophore-sis-based single tube multiplex PCR (mPCR-CE) was used to analyze the MLVA types of Mp in those sam-ples. Results A2063G mutation was identified in 57 specimens including 49 from Beijing,seven from the United States and one from Australia. The 143 Mp-positive specimens were typed into 10 distinct MLVA types. Fifty-four specimens collected in Beijing belonged to four MLVA types, which were M4-5-7-2 (44/54,81.5%),M3-5-6-2 (7/54,13.0%), M4-5-6-2 (2/54,3.70%) and M4-5-5-2 (1/54,1.85%). Fifty-nine specimens collected in the United States belonged to six MLVA types including M4-5-7-2(27/59, 45.8%),M3-5-6-2 (18/59,30.5%),M3-6-6-2 (11/59,18.6%),M3-5-6-1 (1/59,1.69%),M4-5-7-3 (1/59,1.69%) and M5-5-7-2 (1/59,1.69%). Thirty specimens of Mp from Australia were grouped to five types with M3-5-6-2 (12/30, 40.0%) and M4-5-7-2 (10/30, 33.3%) and M3-5-7-2 (5/30, 16.7%) being the predominant types. Macrolide resistance mutations were detected in 57 out of 143 speci-mens (49 from Beijing,seven from the United States and one from Sydney) and 50 of them were MLVA type of M4-5-7-2. Conclusion The MLVA type of M4-5-7-2 is associated with macrolide resistance in Mp.

Isolation of Stenotrophomonas maltophilia from clinical samples: An investigation of patterns motility and production of melanin pigment

Objectives To investigate possible sources of Stenotrophomonas maltophilia (S. maltophilia) in the clinical environment. Methods Different samples were collected from Amol City of Iran. Steps for the identification of S. maltophilia included culturing, biochemical tests, polymerase chain reaction (PCR) of 16S rRNA gene and 23S rRNA gene. In addition, production of melanin pigment and patterns of motility of the bacteria, were also investigated. Results In our study, 20 S. maltophilia strains were isolated from clinical sources, oxygen manometer apparatus of hospitals were 7/110 (6.36%), blood was 1/777 (0.13%), sputum was 4/40 (4%), urine was 1/2 947 (0.03%), tap water was 1/240 (0.42%) and dental suction was 6/120 (5%). The isolated bacteria showed production of melanin pigment with rates of strong, moderate, weak, and lack of pigment. Types of motilities were seen in isolates. Conclusions The highest percentage of bacteria is isolated of oxygen manometer system and dental suction, yet has not been reported from oxygen manometer system. These bacteria have also been associated with patients who have respiratory problems, so it is essential for staffs of hospitals to draw attention to this source of bacteria.

Isolation of Stenotrophomonas maltophilia from clinical samples: An investigation of patterns motility and production of melanin pigment

Objectives:To investigate possible sources of Stenotrophomonas maltophilia (S.maltophilia) in the clinical environment.Methods:Different samples were collected from Amol City of Iran.Steps for the identification of S.maltophilia included culturing,biochemical tests,polymerase chain reaction (PCR) of 16S rRNA gene and 23S rRNA gene.In addition,production of melanin pigment and patterns of motility of the bacteria,were also investigated.Results:In our study,20 S.maltophilia strains were isolated from clinical sources,oxygen manometer apparatus of hospitals were 7/1 10 (6.36％),blood was 1/777 (0.13％),sputum was 4/40 (4％),urine was 1/2947 (0.03％),tap water was 1/240 (0.42％) and dental suction was 6/120 (5％).The isolated bacteria showed production of melanin pigment with rates of strong,moderate,weak,and lack of pigment.Types of motilities were seen in isolates.Conclusions:The highest percentage of bacteria is isolated of oxygen manometer system and dental suction,yet has not been reported from oxygen manometer system.These bacteria have also been associated with patients who have respiratory problems,so it is essential for staffs of hospitals to draw attention to this source of bacteria.

Clinical Characteristics of Macrolide-Resistant Mycoplasma pneumoniae from Children in Jeju

Mycoplasma pneumoniae is the major pathogen of community-acquired pneumonia in children. The prevalence of macrolide-resistant M. pneumoniae (MRMP) is important owing to the limited alternative therapies for children. We analyzed 111 M. pneumoniae obtained from 107 children admitted for lower respiratory tract infection at Jeju National University Hospital between 2010 and 2015. Macrolide resistance of M. pneumoniae was searched for using polymerase chain reaction (PCR) and sequencing. Of 107 clinical M. pneumoniae, 11 (10.3%) carried macrolide resistance mutations in the 23S rRNA gene. All macrolide resistance mutations were A2063G transitions. We found an acquired A2063G mutation of M. pneumoniae from a patient during macrolide treatment. Patients' characteristics and clinical severity did not differ between those with MRMP and macrolide-sensitive M. pneumoniae, with the exception of frequent pleural effusion in the MRMP group. The prevalence of MRMP (10.3%) in Jeju Island was relatively lower than those of surrounding countries in East Asia. Previous antimicrobial usage and timing of diagnostic test should be considered when determining of macrolide resistance of M. pneumoniae.

Analysis of mutations detection in 23S rRNA gene locus of Mycoplasma pneumoniae among children / 国际检验医学杂志

Objective To understand the mutations of macrolide resistance gene locus (23S rRNA) of Mycoplasma pneumoniae (MP) and its correlation with clinical features .Methods A total of 354 respiratory tract samples were collected from children pa-tients with pneumonia .MP and its mutations in 23S rRNA gene locus were detected by real-time PCR .The children cases of MP positive were divided into the mutation group and non-mutation group .Then the clinical data were compared between the two groups .Results Among 354 respiratory tract samples ,166 cases(46 .9% ) were MP positive ,moreover the mutation of 23S rRNA gene locus existed in 135 MP positive samples with the positive detection rate of 81 .3% ,while no 23S rRNA gene locus mutations were detected in 31 samples .Analyzing the clinical data of the mutation group and non-mutation group found that there was no sta-tistical difference in the aspects of age and gender between the two groups .The occurrence rates of severe pneumonia and extrapul-monary complications in the mutation group were higher than those in the non-mutation group (P<0 .05) ,moreover the average hospitalization time and fever duration in the mutation group were longer than those in the non-mutation group (P<0 .05) .Conclu-sion 23S rRNA gene locus mutation has higher detection rate ,prompting that MP shows high resistant rate to macrolides ,which could provide a certain basis for treatment of M P infections .

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.

Diagnosis of Mycoplasma hyorhinis infection in pigs by PCR amplification of 16S-23S rRNA internal transcribed spacer region / 동물의과학연구지

Mycoplasma hyorhinis (M. hyorhinis) is considered an etiological agent of arthritis in suckling pigs. Recently, some M. hyorhinis strains were shown to produce pneumonia that is indistinguishable from the mycoplasmosis caused by M. hyopneumoniae. In this study, we developed a sensitive and specific PCR assay to detect M. hyorhinis and applied the developed PCR assay for detection of Mycoplasma infection in clinical piglets infected with M. hyorhinis. We developed a new PCR assay using a M. hyorhinis-specific primer pair, Mrhin-F and Mrhin-R, designed from the Mycoplasma 16S-23S rRNA internal transcribed spacer (ITS) region. The primers and probe for the assay were designed from regions in the Mycoplasma 16S-23S rRNA ITS unique to M. hyorhinis. The developed PCR assay was very specific and sensitive for the detection of M. hyorhinis. The assay could detect the equivalent of 1 pg of target template DNA, which indicates that the assay was very sensitive. In addition, M. hyorhinis PCR assay detected only M. hyorhinis and not any other Mycoplasma or bacterial spp. of other genera. The new developed PCR assay effectively detected M. hyorhinis infection in pigs. We suggest that this PCR assay using a M. hyorhinis-specific primer pair, Mrhin-F and Mrhin-R, could be useful and effective for monitoring M. hyorhinis infection in pigs.

Detección de Mycoplasma canadense y Mycoplasma californicum en ganado lechero de Argentina / Detection of Mycoplasma canadense and Mycoplasma californicum in dairy cattle from Argentina

Diferentes especies del género Mycoplasma pueden afectar al ganado bovino y causar varias enfermedades. La técnica de PCR, secuenciación y posterior análisis de la región ITS 16S-23S ARNr ha mostrado que existe una importante variabilidad interespecies entre Mollicutes. Se realizó la amplificación (región ITS 16S-23S ARNr) de 16 aislamientos sospechosos de corresponder a alguna especie de Mycoplasma, que habían sido obtenidos de muestras de leche provenientes de rodeos lecheros. Catorce de esos aislamientos fueron PCR positivos. Para confirmar la identidad de Mycoplasma bovis, dichos aislamientos fueron evaluados por otra PCR especie-específica. Siete aislamientos dieron un resultado positivo. Los productos de la PCR de la ITS 16S-23S ARNr de un aislamiento identificado como M. bovis y de otros dos aislamientos identificados como no-M. bovis fueron seleccionados al azar, secuenciados y analizados. Las tres secuencias (A, B y C) mostraron 100 % de similitud con cepas de M. bovis, Mycoplasma canadense y Mycoplasma californicum, respectivamente

Different species of Mycoplasma can affect bovine cattle, causing several diseases. PCR sequencing and further analysis of the 16S-23S rRNA ITS region have shown a significant interspecies variability among Mollicutes. Sixteen suspected isolates of Mycoplasma spp. obtained from milk samples from dairy herds were amplified (16S-23S rRNA ITS region). Fourteen out of those 16 suspected Mycoplasma spp. isolates were PCR-positive. To confirm the identity of Mycoplasma bovis, these 14 isolates were tested by another species-specific PCR. Seven of the isolates rendered a positive result. The products of 16S-23S rRNA ITS PCR from one isolate that was identified as M. bovis and from two other isolates, identified as non- M. bovis were randomly selected, sequenced and analyzed. The three sequences (A, B and C) showed 100% similarity with M. bovis, Mycoplasma canadense and Mycoplasma californicum respectively

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.

Analysis of Gene Mutations Associated with Antibiotic Resistance in Helicobacter pylori Strains Isolated from Korean Patients

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.

Analysis of Gene Mutations Associated with Antibiotic Resistance in Helicobacter pylori Strains Isolated from Korean Patients

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.

The Effect of Concomittent Therarpy and Quadruple Therapy for Patients Who Had 23S Ribosomal Ribonucleic Acid Mutated Helicobacter pylori in Daegu and Kyoungpook Area

BACKGROUND/AIMS: Proton pump inhibitor (PPI) and two types of antimicrobial agents, amoxicillin, and clarithromycin have been widely used for the eradication of Helicobacter pylori. However, antibiotic resistant strains has rapidly increased and has emerged as an important factor for eraducation failure. MATERIALS AND METHODS: Patients diagnosed with chronic gastritis, peptic ulcer disease or gastric epithelial neoplasm was examined by H. pylori PCR for mutation at 23S rRNA. Positive H. pylori PCR without 23S rRNA mutation was eradicated by standard triple therapy. Patients with 23S rRNA mutation was eradicated by standard triple therapy or concomittent therapy with amoxicillin, PPI, clarithromycin and metronidazol or quadruple therapy with bismuth, PPI, tetracycline and metronidazol. We evaluated the predictors of eradication failure with regards to 23S rRNA mutation and initial eradication regimen. RESULTS: Nine hundred sixty-one patients were studied. H. pylori PCR was positive in 35.0% of the patients and 23S rRNA mutatation was found in 22.2% of the patients. The eradication rate of H. pylori for the A2143G point mutated group with standard triple therapy was 28.5% and significantly lower than 93.1% of the wild type group and 100% of the concomitant therapy group, 66.6% of one week quadruple group and 100% of two week quadruple group (P<0.005). CONCLUSIONS: When 23S rRNA point mutation was positive, the standard triple therapy was not effective and the eradication rates was only 22.2%. Alternative regimens should be considered when 23S rRNA point mutation is detected, especially when A2143G point mutation is detected because A2143G point mutation is highly related to eradication failure.

Erythromycin resistance in propionibacteria isolated from patients with acne in Wuhan city is associated with mutations in 23S rRNA gene and presence of erm genes / 中华皮肤科杂志

Objective To determine whether erythromycin-resistant propionibacteria isolated from patients with acne in Wuhan city harbor 23S rRNA gene mutations as well as the transposon Tn5432 carrying ermX genes.Methods Twenty-nine Propionibacterium strains isolated from outpatients with acne in Wuhan city were included in this study.The E-test method was used to determine the susceptibility of these strains to erythromycin and clindamycin.PCR was performed to amplify the 23S rRNA,ermX,ermX (cj),IS1249a and IS1249b genes from resistant strains followed by DNA sequencing and nucleotide alignment.Results Among the 29 Propionibacterium strains,19 were identified as P.acnes and 10 as P.avidum.All of these Propionibacterium strains were resistant to erythromycin (MIC ＞ 256 μg/ml) and clindamycin (MIC ＞ 256 μg/ml),except for 3 P.acnes strains sensitive to clindamycin.Seven P.acnes strains resistant to both antibiotics exhibited an A→G transition at a position cognate with Escherichia coli 23S rRNA 2058.An A→G transition at a position cognate with E.coli 23S rRNA 2059 was identified in one clindamycin-resitant and three clindamycin-sensitive P.acnes isolates.The ermX gene was found in the remaining 8 P.acnes isolates and 2 P.avidum isolates,with the sequence 100％ identical to the reference sequence of the ermX gene of P.acnes in Genbank.Meanwhile,the ermX (cj) gene was successfully amplified from the other 8 P.avidum isolates,which showed 99％ sequence homology with the ermX (cj) gene of Corynebacterium jeikeium,but 94％ homology with the ermX gene of P.acnes in Genbank.Both IS1249a and IS1249b genes were amplified in the 10 ermX gene-positive Propionibacterium strains,but not in the other ermX gene-negative strains.Conclusions The erythromycin resistance in Propionibacterium isolates from Wuhan city may be associated with the A→G transition at the E.coli equivalent bases 2058 and 2059 of the 23S rRNA gene,as well as the presence of the erm X (transferred through the transposon Tn5432) and ermX (c j) genes.

Two Cases of Mycoplasma pneumoniae Pneumonia with A2063G Mutation in the 23S rRNA Gene in Siblings

We describe 2 cases of pneumonia caused by the same macrolide-resistant Mycoplasma pneumoniae in siblings. M. pneumoniae was identified using real-time PCR. Direct sequence analysis of the 23S rRNA gene revealed a point mutation in V domain (A2063G) of the 23S rRNA gene.

Clarithromycin-resistant Helicobacter pylori Associated with 23S rRNA Point Mutations in Jeju Island / 대한소화기학회지

BACKGROUND/AIMS: The point mutations in 23S rRNA gene accounts for the majority of the clarithromycin resistance of Helicobacter pylori. This study aimed to investigate the association between the clarithromycin-resistance of H. pylori and the failure of primary H. pylori eradication therapy in Jeju Island. METHODS: Between April 2011 and October 2012, 6,937 patients underwent endoscopy, and H. pylori infection was evaluated in 2,287 patients (33.0%). Total of 110 patients with H. pylori infection were treated with proton pump inhibitor (PPI)-based triple therapy. The result of eradication was evaluated with urea breath test, histology and PCR which were conducted 4 weeks from the last dose of medicine. RESULTS: The patients who had point mutations were 33 (26.0%). A2142G and A2143G mutations were observed in 10 patients (7.9%) and 23 patients (18.1%). Among 110 patients treated with PPI-based triple therapy, the success rate of the eradication therapy was 52.7% (58/110) and 70.7% (58/82) by intention-to-treat and per-protocol analysis, respectively. Fifteen of the 24 patients who failed the eradication therapy showed point mutations; 1 patient (4.2%) showed A2142G mutation and 14 patients (58.3%) showed A2143G mutation. Patients with A2143G mutation H. pylori showed higher failure rate of 87.5%. Patients with A2142G mutation H. pylori showed similar failure rate compared to those of the patients with wild type H. pylori. CONCLUSIONS: In Jeju Island, the frequency of 23S rRNA point mutations is similar (26.0%) with other regions of Korea (15.8-31.3%). A2143G mutation is associated with the failure of H. pylori eradication.

Macrolide Resistance of Mycoplasma pneumoniae and Its Detection Rate by Real-Time PCR in Primary and Tertiary Care Hospitals

BACKGROUND: This study aimed to evaluate the prevalence of Mycoplasma pneumoniae in primary and tertiary care hospitals and its macrolide resistance rate. METHODS: Nasopharyngeal swabs were collected from 195 pediatric patients in primary and tertiary care hospitals from October to November 2010. The AccuPower MP real-time PCR kit (Bioneer, Korea) was used for the detection of M. pneumoniae. Direct amplicon sequencing was performed to detect point mutations conferring resistance to macrolides in the 23S rRNA gene. RESULTS: Among the 195 specimens, 17 (8.7%) were M. pneumoniae positive, and 3 of the strains (17.6%) obtained from these 17 specimens displayed the A2063G mutation in 23S rRNA. Three macrolide-resistant M. pneumoniae isolates were isolated from patients hospitalized at the primary care hospital. The positive rates of M. pneumoniae for the primary and tertiary care hospitals were 12.1% (15/124) and 2.8% (2/71), respectively (P=0.033). CONCLUSIONS: The positive rate of M. pneumoniae in the primary care hospital was higher than that in the tertiary care hospital. Simultaneous detection of M. pneumoniae and macrolide-resistant mutation genes in the 23S rRNA by real-time PCR is needed for rapid diagnosis and therapy of M. pneumoniae infections.

Linezolid resistance mechanisms and molecular epidemiology of clinical isolates of methicillin-resistant coagulase-negative staphylococci / 中华微生物学和免疫学杂志

Objective To investigate the linezolid resistance mechanisms and molecular epidemiology of clinical isolates of methicillin-resistant coagulase-negative staphylococci (MRCoNS).Methods Seventeen MRCoNS,including 10 S.capitis,4 S.cohnii,2 S.haemolyticus,and 1 S.sciuri with various levels of linezolid resistance were isolated from intensive care units in our hospital from March to August 2011. Minimal inhibitory concentration (MIC) was determined by E-test method. Pulsed-field gel electrophoresis was performed to analyze the molecular epidemiology.PCRs and DNA sequencing were preformed to investigate the mechanisms of linezolid resistance in MRCoNS.Results Nine S.capitis with linezolid MIC of ＞256 μg/ml were indistinguishable,and another S.capitis with linezolid MIC of 4 μg/ml was closely related.Four S.cohnii with linezolid MIC of ＞256 μg/ml were belonged to the same clonal strain.MIC of linezolid for S.sciuri was 64 μg/ml,and were 4 μg/ml and 6 μg/ml for 2 S.haemolyticus,respectively.A commom G2576T mutation and a novel C2104T mutation were identified in 9 S.capitis with linezolid MIC of ＞256 μg/ml by DNA sequence analysis of domain V of the 23S rRNA gene.cfr gene was deteeted in all staphylococci except a S.sciuri whose 23S rRNA gene contained the G2576T mutation.Conclusion It is the first report of linezolid-resistant clinical isolates of staphylococci in China.Linezolid resistance in MRCoNS is related to the presence of DNA mutation in domain V of the 23S rRNA gene and cfr gene.It's a clonally dissemination of linezolid-resistant MRCoNS in intensive care units of our hospital.