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 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.

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 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 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 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.

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.

23S rRNA Gene Mutations Contributing to Macrolide Resistance inCampylobacter jejuniandCampylobacter coli

2009 ◽  
Vol 6 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Scott R. Ladely ◽  
Richard J. Meinersmann ◽  
Mark D. Englen ◽  
Paula J. Fedorka-Cray ◽  
Mark A. Harrison
Keyword(s):  
Gene Mutations ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
23S Rrna Gene

scholarly journals The reduced prevalence of macrolide resistance in Mycoplasma pneumoniae clinical isolates from pediatric patients in Beijing in 2016

2018 ◽  
Author(s):  
Xiujun Tian ◽  
Ran Wei ◽  
Junyan Shao ◽  
Hong Wang ◽  
Jing Li ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Cross Resistance ◽  
Rrna Gene ◽  
Older Children ◽  
Infection Susceptibility ◽  
23S Rrna Gene ◽  
Beijing China ◽  
Different Levels

Older children especially from seven to thirteen years old are more prone to develop Mycoplasma pneumoniae (MP) infection; in winter children are more susceptible to infect with MP. In Beijing, China in 2016 the rates of macrolide resistance of MP were 69.48% (in total children), 61.59% (in outpatients) and 79.28% (in hospitalized patients), respectively. All the macrolide resistant isolates harbored A2063G or A2064G mutation in the 23S rRNA gene. Seven isolates showed a mixed infection. Susceptibility results showed that 73 isolates with the A2063G mutation demonstrated different levels resistance to erythromycin (MIC=8 to>256μg/ml), azithromycin (MIC=8 to>64μg/ml) and josamycin (MIC=2 to 8μg/ml). No cross-resistance was observed in the in the antibiotics of levofloxacin and tetracycline against MP.

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