scholarly journals Detection of Macrolide Resistance in Mycoplasma pneumoniae by Real-Time PCR and High-Resolution Melt Analysis

2008 ◽  
Vol 52 (10) ◽  
pp. 3542-3549 ◽  
Author(s):  
Bernard J. Wolff ◽  
W. Lanier Thacker ◽  
Stephanie B. Schwartz ◽  
Jonas M. Winchell
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
High Resolution Melt ◽  
23S Rrna Gene ◽  
Resistant Strains ◽  
V Region ◽  
Domain V

ABSTRACT Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides or azalides such as erythromycin or azithromycin. Recent studies have discovered the existence of macrolide-resistant strains within the population that have been mapped to mutations within the domain V region of the 23S rRNA gene. Currently, identification of these resistant strains relies on time-consuming and labor-intensive procedures such as restriction fragment length polymorphism, MIC studies, and sequence analysis. The current study reports two distinct real-time PCR assays that can detect the A2063G or A2064G base mutation (A2058G or A2059G by Escherichia coli numbering) conferring macrolide resistance. By subjecting the amplicon of the targeted domain V region of the 23S rRNA gene to a high-resolution melt curve analysis, macrolide-resistant strains can quickly be separated from susceptible strains. Utilizing this method, we screened 100 clinical isolates and found 5 strains to possess mutations conferring resistance. These findings were concordant with both sequencing and MIC data. This procedure was also used successfully to identify both susceptible and resistant genotypes in 23 patient specimens. These patient specimens tested positive for the presence of M. pneumoniae by a separate real-time PCR assay, although the bacteria could not be isolated by culture. This is the first report of a real-time PCR assay capable of detecting the dominant mutations that confer macrolide resistance on M. pneumoniae, and these assays may have utility in detecting resistant strains of other infectious agents. These assays may also allow for clinicians to select appropriate treatment options more rapidly and may provide a convenient method to conduct surveillance for genetic mutations conferring antibiotic resistance.

scholarly journals Antimicrobial Susceptibility of Bordetella pertussis Isolates in Southern Vietnam During 2015–2017

2020 ◽  
Vol 41 (S1) ◽  
pp. s124-s124
Author(s):  
Hoan T. Pham ◽  
Anh H. V. Nguyen ◽  
Cao Huu Nghia
Keyword(s):  
Real Time ◽  
Bordetella Pertussis ◽  
Antimicrobial Susceptibility ◽  
Real Time Pcr ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Southern Vietnam ◽  
Mueller Hinton ◽  
23S Rrna Gene

Background: Pertussis continues to be an important health issue in Vietnam despite infant vaccination programs. In Vietnam, the incidence rates of pertussis per 100,000 population rose from 0.09 in 2014 to 0.33 in 2015 and to 0.58 in 2017. Macrolides, especially erythromycin, are the treatment of choice. However, erythromycin-resistant cases, caused by transition at A2047G position in 23S rRNA, have been reported in the region. Few data are available on antimicrobial resistance in Bordetella pertussis to guide treatment in Vietnam. We report antimicrobial susceptibility of the circulating strains in southern Vietnam during 2015–2017. Methods: Tracheal aspirates from 263 suspected pertussis cases were subject to multiplex real-time PCR to identify B. pertussis and Bordetella spp. Samples were cultured on Regan Lowe agar with 10% sheep blood containing cephalexin (40 µg/mL) and incubated at 37°C for 10 days. The antimicrobial susceptibilities to erythromycin, azithromycin, clarithromycin, and trimethoprim/sulfamethoxazole were determined using the disc diffusion method (CLSI-2017) on Regan Lowe and Mueller Hinton agar. Erythromycin minimum inhibitory concentrations (MICs) were determined using an E-test. The results were recorded after days 3 and 7 of incubation. Sequencing of the 23S rRNA gene was performed to detect mutations conferring macrolide resistance. Results: Of 263 cases, 119 were positive for B. pertussis (45.2%) by real-time PCR, and 15 of 263 strains (5.7%) were successfully cultured. All 15 isolates were susceptible to macrolides and no heterogeneous phenotype was recorded after 7 days; erythromycin MICs were ≤0.094 µg/mL (Fig. 1). We observed no difference in results generated on Regan Lowe and Mueller Hinton media. However, for testing trimethoprim/sulfamethoxazole, results on were superior, as those on Regan Lowe media were unclear. Sequencing of 23S rRNA identified no mutations known to confer macrolide resistance. Conclusions: None of 15 B. pertussis isolates tested were nonsusceptible to erythromycin and macrolides. Similarly, no mutation at the erythromycin-binding site in the 23S rRNA gene was identified. The low isolation rate of B. pertussis by culture means that few positive specimens were tested for antimicrobial susceptibility. To overcome this limitation, detection of resistance directly from clinical specimens needs to be investigated. Ongoing screening for B. pertussis and antimicrobial susceptibility is recommended to support efforts to control the spread of this respiratory tract infection agent.Funding: NoneDisclosures: None

scholarly journals Molecular Basis of Macrolide Resistance inCampylobacterStrains Isolated from Poultry in South Korea

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Bai Wei ◽  
Min Kang
Keyword(s):  
Molecular Mechanisms ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Erythromycin Resistance ◽  
23S Rrna Gene ◽  
Ribosomal Protein L22 ◽  
Resistant Strains ◽  
Azithromycin Resistance

We investigated the molecular mechanisms underlying macrolide resistance in 38 strains ofCampylobacterisolated from poultry. Twenty-seven strains were resistant to azithromycin and erythromycin, five showed intermediate azithromycin resistance and erythromycin susceptibility, and six showed azithromycin resistance and erythromycin susceptibility. FourCampylobacter jejuniand sixCampylobacter colistrains had azithromycin MICs which were 8–16 and 2–8-fold greater than those of erythromycin, respectively. The A2075G mutation in the 23S rRNA gene was detected in 11 resistant strains with MICs ranging from 64 to ≥ 512μg/mL. Mutations including V137A, V137S, and a six-amino acid insertion (114-VAKKAP-115) in ribosomal protein L22 were detected in theC. jejunistrains. Erythromycin ribosome methylase B-erm(B) was not detected in any strain. All strains except three showed increased susceptibility to erythromycin with twofold to 256-fold MIC change in the presence of phenylalanine arginine ß-naphthylamide (PAßN); the effects of PAßN on azithromycin MICs were limited in comparison to those on erythromycin MICs, and 13 strains showed no azithromycin MIC change in the presence of PAßN. Differences between azithromycin and erythromycin resistance and macrolide resistance phenotypes and genotypes were observed even in highly resistant strains. Further studies are required to better understand macrolide resistance inCampylobacter.

scholarly journals Drug Resistance Mechanisms ofMycoplasma pneumoniaeto Macrolide Antibiotics

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Xijie Liu ◽  
Yue Jiang ◽  
Xiaogeng Chen ◽  
Jing Li ◽  
Dawei Shi ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Resistance Mechanisms ◽  
23S Rrna ◽  
Macrolide Antibiotics ◽  
Rrna Gene ◽  
Site Mutation ◽  
23S Rrna Gene ◽  
Resistant Strains ◽  
Domain V ◽  
The 16S Rrna Gene

Throat swabs from children with suspectedMycoplasma pneumoniae(M. pneumoniae) infection were cultured for the presence ofM. pneumoniaeand its species specificity using the 16S rRNA gene. Seventy-sixM. pneumoniaestrains isolated from 580 swabs showed that 70 were erythromycin resistant with minimum inhibitory concentrations (MIC) around 32–512 mg/L. FiftyM. pneumoniaestrains (46 resistant, 4 sensitive) were tested for sensitivity to tetracycline, ciprofloxacin, and gentamicin. Tetracycline and ciprofloxacin had some effect, and gentamicin had an effect on the majority ofM. pneumoniaestrains. Domains II and V of the 23S rRNA gene and the ribosomal protein L4 and L22 genes, both of which are considered to be associated with macrolide resistance, were sequenced and the sequences were compared with the corresponding sequences in M129 registered with NCBI and the FH strain. The 70 resistant strains all showed a 2063 or 2064 site mutation in domain V of the 23S rRNA but no mutations in domain II. Site mutations of L4 or L22 can be observed in either resistant or sensitive strains, although it is not known whether this is associated with drug resistance.

scholarly journals Macrolide-Resistant Mycoplasma pneumoniae in Adults in Zhejiang, China

2014 ◽  
Vol 59 (2) ◽  
pp. 1048-1051 ◽  
Author(s):  
Zibo Zhou ◽  
Xiangzhi Li ◽  
Xiaojian Chen ◽  
Fangjun Luo ◽  
Changwang Pan ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Rflp Analysis ◽  
Zhejiang Province ◽  
23S Rrna ◽  
Rrna Gene ◽  
Type I ◽  
Content Type ◽  
23S Rrna Gene ◽  
Resistant Strains ◽  
Domain V

ABSTRACTMycoplasma pneumoniaeis a major pathogen causing community-acquired pneumoniae (CAP), which is generally treated with macrolides. In recent years, however, although macrolide-resistantM. pneumoniaehas been reported frequently, particularly in China, very little is known about the prevalence of macrolide-resistantM. pneumoniaeinfection in adults. In this study, we survey the macrolide-resistantM. pneumoniaein adults in Zhejiang province and characterize the mechanisms of resistance to macrolide. Six hundred fifty throat swab samples were collected from adult patients with CAP from January 2012 to August 2014. These samples were assayed by nested PCR and then cultivated forM. pneumoniae. All isolates were sequenced to determine the mutation in domain V of the 23S rRNA gene. The activities of 10 antibiotics against macrolide-resistantM. pneumoniaeisolates were also investigatedin vitro. Moreover, restriction fragment length polymorphism (RFLP) analysis of the amplified P1 gene was used to type 50 resistant strains. One hundred percent (71/71) ofM. pneumoniaestrains isolated from adults with CAP were resistant to erythromycin (MIC = 128 to >256 μg/ml), clarithromycin (MIC = 128 to >256 μg/ml), and azithromycin (MIC = 32 to >64 μg/ml). Furthermore, all macrolide-resistantM. pneumoniaestrains identified had an A2063G mutation in domain V of the 23S rRNA gene. Forty-six resistant strains (92.0%) were classified into type I strain on the basis of P1 gene PCR-RFLP analysis. According to these findings, it is suggested that macrolide-resistantM. pneumoniaeinfection is very prevalence among adults in Zhejiang province. Thus, there is necessary to perform the epidemiological monitoring of macrolide-resistantM. pneumoniaein the future.

scholarly journals Ribosomal Mutations as the Main Cause of Macrolide Resistance in Campylobacter jejuni and Campylobacter coli

2011 ◽  
Vol 55 (12) ◽  
pp. 5939-5941 ◽  
Author(s):  
Mirva Lehtopolku ◽  
Pirkko Kotilainen ◽  
Marjo Haanperä-Heikkinen ◽  
Ulla-Maija Nakari ◽  
Marja-Liisa Hänninen ◽  
...  
Keyword(s):  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Resistance Mutations ◽  
Content Type ◽  
Amino Acid Insertion ◽  
23S Rrna Gene ◽  
Ribosomal Protein L22 ◽  
Resistant Strains

ABSTRACTThe aim of this study was to examine macrolide resistance mutations inCampylobacterspecies. In 76 strains studied, point mutation A to G at position 2059 of the 23S rRNA gene was detected in 30 of the 33 erythromycin-resistant strains. An amino acid insertion in the ribosomal protein L22 was found in one resistant strain without a 23S rRNA mutation. The A2059G mutation is the main cause of macrolide resistance inCampylobacterspecies.

scholarly journals Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas

2009 ◽  
Vol 75 (9) ◽  
pp. 2945-2950 ◽  
Author(s):  
Jennifer Hodgetts ◽  
Neil Boonham ◽  
Rick Mumford ◽  
Matthew Dickinson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Multiplex Assay ◽  
The Other ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Detection ◽  
23S Rrna Gene ◽  
Pcr Assays

ABSTRACT Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays.

scholarly journals 961. Prevalence and Macrolide Resistance of Mycoplasma genitalium After Initiation of HIV Preexposure Prophylaxis

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S29-S29
Author(s):  
Jens Van Praet ◽  
Sanne Steyaert ◽  
Stefaan Vandecasteele ◽  
Barbara Van Den Bergh ◽  
Hilde Mahieu ◽  
...  
Keyword(s):  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Sexually Transmitted ◽  
Preexposure Prophylaxis ◽  
23S Rrna Gene ◽  
Resistant Strains ◽  
Taqman Array

Abstract Background Recent evidence shows that patients using HIV preexposure prophylaxis (PrEP) have an increased rate of bacterial sexually transmitted infections (STIs), including syphilis, chlamydia, and gonorrhea. The rate of Mycoplasma genitalium infections and the susceptibility of M. genitalium in patients on PrEP have been less well described. Methods We studied all patients who started on PrEP in the AZ Sint-Jan Hospital Bruges from January 6, 2017 to January 4, 2019. Patients were screened for M. genitalium and other bacterial STIs with rectal swabs, pharyngeal swabs, first-voided urine and blood collections at baseline and quarterly intervals after initiating PrEP. TaqMan array card technology was used to detect M. genitalium and determine macrolide-resistance mediating mutations in the region V of the 23S rRNA gene (A2058G, A2059G, A2058C, and others). Patients with an STI were treated based on a national guideline. Proportions were estimated using a Generalized Estimating Equations model with independent correlation structure. Results A total of 136 males and 1 female (median age, 40 years (interquartile range (IQR), 20–79)) were included in the study. All men were gay or bisexual. The median follow-up time was 11.3 months (IQR, 4.7–15.3). In total, 117 patients (85%) used PrEP daily at their last visit. The estimated proportion of patients with M. genitalium at baseline, 3 months, 6 months, 9 months, and 12 months was 7% (95% CI 4–13), 12% (95% CI 7–20), 7% (95% CI 4–15), 6% (3–15), and 6% (2–15). Thirty-two patients (23%) tested at least once positive for M. genitalium during the study period. The estimated percentage of macrolide resistance increased from 40% (95% CI 16–70) at baseline to 71% (95% CI 44–89), 67% (95% CI 27–92), 80% (95% CI 31–97), and 75% (95% CI 24–97) at 3 months, 6 months, 9 months, and 12 months, respectively. Conclusion After initiation of PrEP, the prevalence of M. genitalium in our cohort at quarterly screening was not increased compared with baseline. However, a nonsignificant trend of an increased percentage of macrolide-resistant strains was observed. Disclosures All Authors: No reported Disclosures.

Identification of a Novel Mutation Affecting Domain V of the 23S rRNA Gene inHelicobacter pylori

Helicobacter ◽  
2004 ◽  
Vol 9 (5) ◽  
pp. 396-399 ◽  
Author(s):  
Sonia Toracchio ◽  
Gitana M. Aceto ◽  
Renato Mariani-Costantini ◽  
Pasquale Battista ◽  
Leonardo Marzio
Keyword(s):  
Novel Mutation ◽  
23S Rrna ◽  
Rrna Gene ◽  
23S Rrna Gene ◽  
Domain V

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.

scholarly journals Intrinsic Macrolide Resistance in Mycobacterium smegmatis Is Conferred by a Novel erm Gene, erm(38)

2003 ◽  
Vol 47 (10) ◽  
pp. 3053-3060 ◽  
Author(s):  
Kevin A. Nash
Keyword(s):  
Mycobacterium Smegmatis ◽  
Sequence Data ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Cross Resistance ◽  
Rrna Gene ◽  
Mycobacterium Fortuitum ◽  
Knockout Mutant ◽  
23S Rrna Gene ◽  
High Level

ABSTRACT High-level, acquired macrolide resistance in mycobacteria is conferred by mutation within the 23S rRNA gene. However, several mycobacteria are naturally resistant to macrolides, including the Mycobacterium smegmatis group and Mycobacterium tuberculosis complex. Thus, the aim of this study was to characterize this resistance. Intrinsic macrolide resistance in M. smegmatis was inducible and showed cross-resistance to lincosamides but not to streptogramin B (i.e., ML resistance). A similar phenotype was found with Mycobacterium microti and macrolide-resistant Mycobacterium fortuitum. A search of the DNA sequence data for M. smegmatis strain mc2155 identified a novel erm gene, erm(38), and expression analysis showed that erm(38) RNA levels increased >10-fold after a 2-h incubation with macrolide. Inducible ML resistance was not expressed by an erm(38) knockout mutant, and complementation of this mutant with intact erm(38) in trans resulted in high-level ML resistance (e.g., clarithromycin MIC of >512 μg/ml). Thus, the results indicate that erm(38) confers the intrinsic ML resistance of M. smegmatis. Southern blot analysis with an erm(38)-specific probe indicated that a similar gene may be present in macrolide-resistant M. fortuitum. This finding, with the presence of the erm(37) gene (Rv1988) in the M. tuberculosis complex, suggests that such genes are widespread in mycobacteria with intrinsic macrolide resistance.

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