Macrolide Resistance-associated Mutations Research Articles

Epidemic Features and Megagenomic Analysis of Childhood Mycoplasma Pneumoniae post COVID-19 Pandemic: A 6-year Study in Southern China

ABSTRACT With the atypical rise of Mycoplasma pneumoniae infection (MPI) in 2023, prompt studies are needed to determine the current epidemic features and risk factors with emerging trends of MPI to furnish a framework for subsequent investigations. This multicentre, retrospective study was designed to analyse the epidemic patterns of MPI before and after the COVID-19 pandemic, as well as genotypes and the macrolide resistance-associated mutations in MP sampled from pediatric patients in Southern China. Clinical data was collected from 133674 patients admitted into investigational hospitals from June 1, 2017, to November 30, 2023. Metagenomic next-generation sequencing (mNGS) data were retrieved based on MP sequence positive samples from 299 pediatric patients for macrolide resistance-associated mutations analysis. Pearson’s chi-squared test was used to compare categorical variables between different time frames. The monthly average cases of pediatric common respiratory infection diseases were increased without enhanced public health measures after the pandemic, especially for influenza, respiratory syncytial virus infection, and MPI. The contribution of MPI to pneumoniae was similar to that in the outbreak in 2019. Compared mNGS data between 2019-2022 and 2023, the severity of MP did not grow stronger despite higher rates of macrolide-resistance hypervariable sites, including loci 2063 and 2064, were detected in childhood MP samples of 2023. Our findings indicated ongoing surveillance is necessary to understand the impact of post pandemic on MP transmission disruption on epidemic season and severity of clinical outcomes in different scenarios.

Chest Computed Tomography Radiomics for Determining Macrolide Resistance-Associated Gene Mutation Status in Children with Mycoplasma pneumoniae Pneumonia: A Two-Center Study

Rationale and ObjectivesThe mutations in the 23S ribosomal RNA (rRNA) gene are associated with an increase in resistance to macrolides in children with Mycoplasma pneumoniae pneumonia (MPP). This study aimed to develop and validate a chest computed tomography (CT) radiomics model for determining macrolide resistance-associated gene mutation status in MPP. Materials and MethodsA total of 258 MPP patients were retrospectively included from two institutions (training set: 194 patients from the first institution; external test set: 64 patients from the second). The 23S rRNA gene mutation status was tested by nasopharyngeal swab polymerase chain reaction. Radiomics features were extracted from chest CT images of pulmonary lesions segmented with semi-automatic delineation. Subsequently, radiomics feature reduction was applied to identify the most relevant features. Logistic regression and random forest algorithms were employed to establish the radiomics models, which were five-fold cross-validated in the training set and validated in the external test set. ResultsThe radiomics feature selection resulted in eight features. After five-fold cross-validation in the training set, the mean areas under the receiver operating characteristic curve (AUCs) of the logistic regression and random forest models were 0.868 (95% confidence interval (CI): 0.813-0.923) and 0.941 (95% CI: 0.907-0.975), respectively. In the external test set, the corresponding AUCs were 0.855 (95% CI: 0.758-0.952) and 0.815 (95% CI: 0.705-0.925). ConclusionChest CT radiomics is a promising diagnostic tool for determining macrolide resistance gene mutation status in MPP. Availability of data and materialThe datasets generated or analyzed during the study are available from the corresponding author on reasonable request.

Estimation of antimicrobial resistance of Mycoplasma genitalium, Belgium, 2022.

BackgroundAntimicrobial resistance (AMR) of Mycoplasma genitalium (MG) is a growing concern worldwide and surveillance is needed. In Belgium, samples are sent to the National Reference Centre of Sexually Transmitted Infections (NRC-STI) on a voluntary basis and representative or robust national AMR data are lacking.AimWe aimed to estimate the occurrence of resistant MG in Belgium.MethodsBetween July and November 2022, frozen remnants of MG-positive samples from 21 Belgian laboratories were analysed at the NRC-STI. Macrolide and fluoroquinolone resistance-associated mutations (RAMs) were assessed using Sanger sequencing of the 23SrRNA and parC gene. Differences in resistance patterns were correlated with surveillance methodology, socio-demographic and behavioural variables via Fisher's exact test and logistic regression analysis.ResultsOf the 244 MG-positive samples received, 232 could be sequenced for macrolide and fluoroquinolone RAMs. Over half of the sequenced samples (55.2%) were resistant to macrolides. All sequenced samples from men who have sex with men (MSM) (24/24) were macrolide-resistant. Fluoroquinolone RAMs were found in 25.9% of the samples and occurrence did not differ between socio-demographic and sexual behaviour characteristics.ConclusionAlthough limited in sample size, our data suggest no additional benefit of testing MG retrieved from MSM for macrolide resistance in Belgium, when making treatment decisions. The lower occurrence of macrolide resistance in other population groups, combined with emergence of fluoroquinolone RAMs support macrolide-resistance testing in these groups. Continued surveillance of resistance in MG in different population groups will be crucial to confirm our findings and to guide national testing and treatment strategies.

Mycoplasma genitalium infection among gay, bisexual and other men who have sex with men in Montréal, Canada.

The bacteria Mycoplasma genitalium has been identified as a causative agent of urethritis in men, especially in gay, bisexual and other men who have sex with men (gbMSM). Canadian clinic-based data have identified a high prevalence of M. genitalium and resistance to antibiotic treatments. This article estimates the prevalence of M. genitalium infections among Montréal gbMSM, explores correlates for M. genitalium infection and estimates the prevalence of mutations associated with antimicrobial resistance (AMR). Engage Cohort Study is a multi-site longitudinal study on sexually active gbMSM, aged 16 years and older, recruited via respondent-driven sampling in Montréal, Toronto and Vancouver. Participants completed a questionnaire on behaviour and were tested for sexually transmitted and blood-borne infections at each visit. For this sub-study, Montréal participants with a follow-up visit that occurred between November 2018 and November 2019 were included. A total of 2,064 samples were provided by 716 participants. Prevalence of M. genitalium infection was 5.7% at rectal and/or urethral sites, 4.0% at rectal site and 2.2% at urethral site. Correlates for M. genitalium infection were younger age and reporting six or more sexual partners in the past six months. Prevalence of macrolide resistance associated mutations (MRAM), quinolone resistance associated mutations (QRAM) and either MRAM or QRAM, was 82%, 29% and 85%, respectively. This first population-based study among gbMSM in Canada documents a high prevalence of urethral and rectal M. genitalium infection and high levels of AMR. Our results highlight the importance of access to testing and AMR detection when indicated.

2662. Efficacy of Sitafloxacin and Doxycycline Combination Therapy as a Salvage Treatment for Refractory Mycoplasma genitalium Infection

Abstract Background The prevalence of resistant strains of Mycoplasma genitalium has increased over the past few decades, leading to limited treatment options. This study aimed to evaluate the efficacy of sitafloxacin and doxycycline combination therapy as a salvage treatment for refractory rectal and urogenital M. genitalium infections. Methods Men who have sex with men (MSM) diagnosed to have M. genitalium infection from urine samples or rectal swabs using a PCR assay were evaluated and administered sitafloxacin 200 mg and doxycycline 200 mg daily for 21 days. Each sample was tested for 23rRNA, parC, and gyrA mutations before and if a failure after the treatment. Results A total of 27 MSMs were included. The median age was 33 years (range 21-47), and 33.3% (9/27) were people with HIV(PWH) (Table). Among PWH, the median CD4 count was 642, and all were virally suppressed with antiretroviral therapy. Positive samples included 22 rectal and 5 urine specimens. The time to test of cure(TOC) was 14 days (range 0-227). In the two failure cases 9 and 20, the TOC was conducted too early, and it was rechecked after 21 days following treatment completion; both rechecked TOCs were positive. ParC mutations were detected in 100%(23/23) of the samples, gyrA mutations in 66.7%(16/24) and ,macrolide-resistance associated mutations in 100%(22/22). Among the parC mutations, 95.7%(22/23) was G248T(S83I), while 4.3%(1/23) was G259T(D87Y). The overall cure rate was 81.5%. The cure for rectal infection was 81.8%(18/22), while the cure rate for urogenital infection was 80%(4/5). No significant difference was observed between the anatomical sites (p=0.924). The cure rate for strains harboring parC mutation and wild-type in gyrA was 85.7% (6/7), whereas the cure rate for the strain harboring parC mutation and gyrA mutation was 73.3%(11/15). No significant difference was found regardless of gyrA mutations (p=0.519). Additionally, no significant difference was observed in the cure rate between TOC < 21 days and >= 21 days (p=0.964). Cases of Mycoplasma genitalium treatment and antimicrobial-resistance profiles in participants before and after treatment with the combination therapy Conclusion The combination therapy showed high efficacy of 81.5% for highly resistant M. genitalium infection and can be considered as a salvage treatment option for refractory cases. Disclosures All Authors: No reported disclosures

Molecular typing of Mycoplasma pneumoniae and its correlation with macrolide resistance in children in Henan of China

Background/purposeAs a major causative pathogen of community-acquired pneumonia, Mycoplasma pneumoniae (M. pneumoniae) can cause both upper and lower respiratory tract inflammation as well as extrapulmonary syndromes, especially in infants and the elderly. The emergence of macrolide–resistance has significant effects on the treatment of relevant diseases in children. This study aimed to analyze the genotypes and the macrolide resistance-associated mutations in M. pneumoniae sampled from the pediatric patients in Henan, China. MethodsA segment of gene on the 23S rRNA was amplified and sequenced to detect the mutations related to macrolide resistance. Molecular typing was performed by the method named multiple locus variable-number tandem repeat analysis (MLVA) for macrolide-susceptible and macrolide-resistant specimens. ResultsAmong the M. pneumoniae-positive samples, 95.7% (111/116) had macrolide-resistant mutation, and all of them consisted of the A2063G mutation. There were only two MLVA types identified in this study, type 4-5-7-2 (51/92, 55.4%) and type 3-5-6-2 (41/92, 44.6%). ConclusionThere was no correlation between MLVA types and macrolide resistance (P ​> ​0.05).

Retrospective Evaluation of Mycoplasma genitalium Prevalence and Macrolide Resistance in a Study Cohort of Pregnant Women in Birmingham, AL, From 1997 to 2001.

In a prospective study conducted in 2020 to 2021, macrolide resistance-associated mutations were found in 41% of pregnant persons in Birmingham, AL, with Mycoplasma genitalium detected. We retrospectively evaluated M. genitalium in 203 pregnant persons participating in a study conducted in 1997 to 2001 in Birmingham and adjacent areas and found a prevalence of 11% (95% confidence interval, 6.9%-15.7%), but no macrolide resistance-associated mutations.

Phenotypic and Genotypic Antimicrobial Susceptibility Testing of Chlamydia trachomatis Isolates from Patients with Persistent or Clinical Treatment Failure in Spain

The aim of this multicentre project (seven hospitals across the Spanish National Health Service) was to study the phenotypic and genotypic susceptibility of C. trachomatis to the main antimicrobials used (macrolides, doxycycline, and quinolones) in isolates from patients with clinical treatment failure in whom reinfection had been ruled out. During 2018–2019, 73 clinical isolates were selected. Sixty-nine clinical specimens were inoculated onto confluent McCoy cell monolayers for phenotypic susceptibility testing. The minimum inhibitory concentration for azithromycin and doxycycline was defined as the lowest concentration associated with an at least 95% reduction in inclusion-forming units after one passage in the presence of the antibiotic compared to the initial inoculum for each strain (control). Sequencing analysis was performed for the genotypic detection of resistance to macrolides, analysing mutations in the 23S rRNA gene (at positions 2057, 2058, 2059, and 2611), and quinolones, analysing a fragment of the gyrA gene, and searching for the G248T mutation (Ser83->Ile). For tetracyclines, in-house RT-PCR was used to test for the tet(C) gene. The phenotypic susceptibility testing was successful for 10 isolates. All the isolates had minimum inhibitory concentrations for azithromycin ≤ 0.125 mg/L and for doxycycline ≤ 0.064 mg/L and were considered sensitive. Of the 73 strains studied, no mutations were found at positions T2611C or G248T of the gyrA gene. We successfully sequenced 66 isolates. No macrolide resistance-associated mutations were found at positions 2057, 2058, 2059, or T2611C. None of the isolates carried the tet(C) gene. We found no evidence for genomic resistance in this large, clinically relevant dataset.

Retrospective and Comparative Study of Three Molecular Assays for the Macrolide Resistance Detection in Mycoplasma genitalium Positive Urogenital Specimens.

The capacity of Mycoplasma genitalium to develop resistance to macrolides makes detection of macrolide resistance genes by rapid real-time PCR assays increasingly necessary in clinical diagnostic laboratories so as to initiate appropriate treatment as rapidly as possible. The aim of this retrospective and comparative study was to conduct the clinical evaluation of three commercially available kits for macrolide resistance detection. A total of 111 M. genitalium positive samples analyzed in the Clinical Microbiology Laboratory of the Miguel Servet University Hospital, Zaragoza (Spain) were used. After M. genitalium molecular confirmation, the three assays under study were evaluated and discrepant results were resolved via sequencing. The clinical sensitivity for resistance detection was 83% (95% confidence interval, 69% to 93%) for the ResistancePlus® MG panel kit (SpeeDx Pty Ltd., Sydney, Australia), 95% (84% to 99%) for AllplexTM MG & AziR Assay (Seegene®, Seoul, Korea), and 97% (88% to 99%) for the VIASURE macrolide resistance-associated mutations (23SrRNA) Real time PCR detection kit (Certest Biotec, Zaragoza, Spain). The clinical specificity was 100% (94% to 100%) for Allplex and VIASURE assays and 95% (86% to 99%) for SpeeDx assay. The results arising from this study are cause for strong consideration for the implementation of rapid real-time PCR assays in clinical diagnosis laboratories to eliminate treatment failure and transmission as soon as possible.

Molecular Characteristics of Macrolide Resistance in Treponema pallidum from Patients with Latent Syphilis in Xinjiang, China.

Macrolide resistance in Treponema pallidum (T. pallidum) has been increasing in recent years worldwide. However, few data are available on macrolide resistance in T. pallidum from Xinjiang province, located in the western part of China, which is an area with a high incidence of syphilis. In this study, we investigated the molecular characteristics of macrolide resistance in T. pallidum from patients with latent syphilis in Xinjiang, China. In total, 204 whole blood samples were collected from patients with latent syphilis during 2016 to 2017, in the First Hospital of Xinjiang Medical University. Genomic DNA of blood samples was extracted using a QIAamp DNA Mini Kit and T. pallidum was detected by PCR with the specific polA gene of T. pallidum. The 23S rRNA gene of T. pallidum was amplified among the T. pallidum-positive samples by nested PCR, and macrolide resistance-associated mutation sites A2058G and A2059G in the 23S rRNA gene were identified using restriction enzymes MboII and BsaI. The specific polA gene of T. pallidum (T. pallidum positive) was detected in 27 blood samples (13.2%) from 204 patients with latent syphilis. The 23S rRNA gene was then amplified in all 27 T. pallidum-positive samples, among which 24/27 samples (88.9%) harbored the A2058G mutation in the 23S rRNA gene and 3/27 (11.1%) had the A2059G mutation. Our results indicated that T. pallidum macrolide resistance should not be ignored in Xinjiang, China, and that A2058G was the predominant macrolide resistance mechanism. Blood may be a suitable specimen for the detection of resistant mutations of T. pallidum in patients with latent syphilis who do not show any clinical symptoms.

Prevalence of fluoroquinolone resistance-associated mutations in Mycoplasma genitalium among clients of two sexual health centres in the Netherlands: a cross-sectional study

ObjectiveThis study aimed to determine the prevalence of fluoroquinolone resistance-associated mutations (QRAMs) in Mycoplasma genitalium (MG) among clients of two sexual health centres (SHCs) in the Netherlands.DesignA cross-sectional study.Setting and...

Emerging multidrug resistance contributes to treatment failure in Mycoplasma genitalium infected patients in China

The increased treatment failure in patients with sexually transmitted Mycoplasma genitalium infection is associated with antimicrobial resistance. We performed a retrospective survey of antimicrobial clinical outcomes in M. genitalium-infected patients. In addition, we detected macrolide, fluoroquinolone and tetracycline resistance-associated markers to determine their role in treatment failure. The overall incidence of treatment failure was 28.29%, regardless of the drug used. In the present study, the prevalence of macrolide resistance-associated mutations in the 23S rRNA gene was 64%; that of fluroquinolone resistance-associated S83/D87 substitutions in the parC gene was 67.5%; and that of the tetracycline resistance-associated C1192T mutation in the 16S rRNA gene was 22.5%. Furthermore, evidence of dual antimicrobial resistance was found in 46.4% of pretreatment samples, and triple antimicrobial resistance was identified in 9.9%. These findings regarding the emergence of multidrug-resistant strains of M. genitalium in China are concerning and emphasize the importance of guiding M. genitalium treatment with antimicrobial resistance assays.

Mycoplasma genitalium and Antimicrobial Resistance Among a Cohort of West African Men Who Have Sex With Men Using Preexposure Prophylaxis (CohMSM-PrEP ANRS 12369-Expertise France Study).

Antimicrobial resistance to macrolides and fluoroquinolones in Mycoplasma genitalium (MG) among men who have sex with men (MSM) is worryingly high in high-resource countries. Data in Africa are lacking. We aimed to assess the burden of MG including the presence of resistance-associated mutations (RAMs) in MG among MSM using human immunodeficiency virus preexposure prophylaxis in Burkina Faso, Côte d'Ivoire, Mali, and Togo. MSM were included in a prospective cohort study (2017-2021). Molecular detection of MG in urine, anorectal, and pharyngeal samples was performed at baseline and after 6 and 12 months. Detection of RAMs to macrolides and fluoroquinolones was performed by sequencing the 23S ribosomal RNA, parC, and gyrA genes. A sample was found to be possibly resistant to fluoroquinolones if alterations were found in ParC position 83/87. Of 598 participants, 173 (28.9%) were positive at least once for MG and global point-prevalence was 19.4%. Interestingly, 238 of 250 (95.2%) infections were asymptomatic and 72 of 138 MG infections with follow-up data (52.2%) cleared during the study. Only 1 macrolide RAM was found (0.6%). Prevalence of fluoroquinolones RAMs was 11.3% overall, ranging from 2.4% in Burkina Faso to 17.5% in Mali. Although MG was highly prevalent in these MSM, macrolide resistance was almost nonexistent. Nevertheless, >10% of the samples were possibly resistant to fluoroquinolones. Heterogeneity in the prevalence of fluoroquinolone RAMs between countries may be explained by different antimicrobial consumption in humans and animals.

Low prevalence of macrolide resistance in Mycoplasma genitalium infections in a cohort of pregnant women living with human immunodeficiency virus.

Macrolide resistance in Mycoplasma genitalium (M. genitalium) is increasing as a result of the widespread use of azithromycin in the treatment of sexually transmitted infections (STIs). To date, there are few published studies on macrolide resistance patterns in South African pregnant women. This study now contributes to the growing body of knowledge. This study included 385 pregnant women living with HIV. Vaginal swabs were collected from consenting pregnant women and used for the detection of M. genitalium using the TaqMan assay. Macrolide resistance-associated mutations in the 23S rRNA gene were determined for all samples that tested positive for M. genitalium using the AllplexTM MG & AziR assay (Seegene) which allows for the simultaneous detection and identification of M. genitalium and six mutations (A2058C, A2058G, A2058T, A2059C, A2059G and A2059T) responsible for azithromycin resistance. The correlation between the TaqMan assay and AllplexTM MG & AziR assay (Seegene) for the detection of M. genitalium was also performed in a subset of 121 samples. Of the 385 samples tested in this study, 14 samples were positive for M. genitalium estimating a prevalence of 3.6%. The same 14 samples also tested positive on the AllplexTM assay indicating a good correlation between the TaqMan Assay and the AllplexTM. Of the 14 positive samples, one sample carried a mutation at position A2059G denoting macrolide resistance in this pathogen. Mutations in the other regions of the 23S rRNA were not detected. All assay controls used in the mutation scanning produced the desired results showing the validity of the assay. In this study, macrolide resistance in M. genitalium was detected. Despite the low prevalence of resistance determinants ongoing antimicrobial resistance surveillance is vital considering that azithromycin is used in the syndromic management for the treatment of vaginal discharge syndrome.

High frequency of Nichols-like strains and increased levels of macrolide resistance in Treponema pallidum in clinical samples from Buenos Aires, Argentina

Globally, 94% of Treponema pallidum subsp. pallidum (TPA) clinical strains belong to the SS14-like group and 6% to the Nichols-like group, with a prevalence of macrolide resistance of 90%. Our goal was to determine whether local TPA strain distribution and macrolide resistance frequency have changed significantly since our last report, which revealed that Buenos Aires had a high frequency of Nichols-like strains (27%) and low levels of macrolide resistance (14%). Swab samples from patients with suspected syphilis were collected during 2015–2019 and loci TP0136, TP0548, TP0705 were sequenced in order to perform multilocus sequence typing. Strains were classified as Nichols-like or SS14-like. The presence of macrolide resistance-associated mutations was determined by examination of the 23S rDNA gene sequence. Of 46 typeable samples, 37% were classified as Nichols-like and 63% as SS14-like. Macrolide resistance prevalence was 45.7%. Seven allelic profiles were found, five were SS14-like and two were Nichols-like. The frequency of Nichols-like strains increased between studies (26.8% vs. 37%, p = 0.36). A dramatic increase was found in the frequency of macrolide resistant strains between studies (14.3% vs. 45.7%, p = 0.005). Our results are in agreement with international trends and underscore the need to pursue further TPA molecular typing studies in South America.

Prevalence of macrolide and fluoroquinolone resistance-associated mutations in Mycoplasma genitalium in metropolitan and overseas France

ObjectiveLimited macrolide and fluoroquinolone resistance data are available in France for Mycoplasma genitalium. We performed a multicentre cross-sectional study to investigate the prevalence of macrolide and fluoroquinolone resistance-associated mutations in...

Molecular Typing Reveals Distinct Mycoplasma genitalium Transmission Networks among a Cohort of Men Who Have Sex with Men and a Cohort of Women in France.

Mycoplasma genitalium causes sexually transmitted infecti.ons in men and women. Treatment failures to macrolides and fluoroquinolones have been reported worldwide. Although the mgpB typing method has often been used in M. genitalium-infected men who have sex with men (MSM), limited typing data are available for M. genitalium-infected women. In this study, we aimed to investigate the genetic relationship between M. genitalium strains and their antibiotic resistance profile in a cohort of MSM (86.2% on HIV preexposure prophylaxis [PrEP], 13.8% HIV positive) and a large cohort of women using mgpB/MG309 typing. The mgpB types were determined in 374 samples from 305 women and 65 MSM. Three MSM and one woman had two concurrent or subsequent samples. Macrolide and fluoroquinolone resistance-associated mutations were searched in the 23S rRNA as well as parC and gyrA genes. The mgpB phylogenetic construction revealed three large clusters that differed according to sexual practices and geographical origin of patients. The prevalence of macrolide and fluoroquinolone resistance was significantly higher in MSM compared with women (95.4% vs. 14.1% and 30.6% vs. 7.2%, p < 0.001, respectively). The macrolide resistance spread was polyclonal in both populations, but clonal diffusion of two dual-resistant types was observed in PrEP users in association with high antibiotic pressure and dense connectivity in this population.

Detection of highly macrolide-resistant Legionella pneumophila strains from a hotel water network using systematic whole-genome sequencing.

Implementation of an antibiotic resistance detection tool in Legionella daily surveillance at the French National Reference Centre for Legionella. Systematic WGS of Legionella pneumophila isolates and bioinformatics detection of specific mutations linked to antibiotic resistance. Phenotypic validation of antibiotic resistance detected by WGS was performed by the broth microdilution method. More than 3000 L. pneumophila strains were screened for antibiotic resistance. A macrolide resistance-associated A2052G mutation in the 23S rRNA gene was identified in the genome of eight isolates from a hotel water network. High-level macrolide resistance (i.e. MICs of 1024-2048 mg/L for azithromycin and erythromycin) with no cross-resistance to other antimicrobials was phenotypically confirmed by antimicrobial susceptibility testing for the eight isolates. Systematic WGS of L. pneumophila is a powerful tool for first-line high-throughput screening of antibiotic resistance before phenotypic validation.

Direct assessment of possible mutations in the 23S rRNA gene encoding macrolide resistance in Chlamydia trachomatis.

Reports of potential treatment failure have raised particular concerns regarding the efficacy of the single dose azithromycin regimen in the treatment of urogenital and anorectal Chlamydia trachomatis (CT) infections. Several factors have been suggested, including heterotypic resistance. Antimicrobial susceptibility testing in CT requires cell culture with serial dilutions of antibiotics, which is laborious and for which there is no standardized testing methodology. One method to partly overcome these difficulties would be to use a genotypic resistance assay, however most current available assays do still require prior CT culture. In order to facilitate the assessment of genotypic resistance directly from clinical samples, without the need for prior culture, the aim of this study was to develop a CT specific PCR assay for the assessment of resistance associated mutations (RAMs) in the 23S rRNA gene, and to evaluate a sample of clinical cases in which CT PCR's remained positive during follow-up despite azithromycin treatment. Neither the in silico analysis nor the analytical specificity testing demonstrated clinically relevant cross-reactivity with other bacterial species. These results in conjunction with the analytical sensitivity demonstrating consistent CT 23S rRNA gene detection in the range of 10e3 IFU/mL, exemplify the assay's apt performance. Although no known macrolide RAMs were detected in the clinical cases, the described assay allows future culture independent macrolide RAM surveillance in CT, and increases accessibility for other laboratories to engage in screening.

Molecular Epidemiological Characteristics of Mycobacterium abscessus Complex Derived from Non-Cystic Fibrosis Patients in Japan and Taiwan.

ABSTRACTMycobacterium abscessus complex (MABC) is a group of emerging, highly antimicrobial-resistant non-tuberculous mycobacteria. Specific MABC clones are spreading globally in patients with cystic fibrosis (CF); however, associated genomic epidemiology is lacking in East Asia, with very few patients with CF. Here, we investigated MABC populations derived from non-CF patients in Japan and Taiwan. Analysis of whole-genome sequencing data of 220 MABC isolates revealed that 112, 105, and 3 were M. abscessus subsp. abscessus (ABS), M. abscessus subsp. massiliense (MAS), and M. abscessus subsp. bolletii (BOL), respectively. Moreover, >50% of ABS and >70% of MAS were related to four predominant clones in the region. Known mutations conferring macrolide resistance were rare (1.4%) and were not enriched in the predominant clones. Conversely, the macrolide-susceptible erm(41) T28C mutation was significantly enriched in one predominant ABS clone. The most predominant ABS clone was genetically related to the previously described dominant circulating clone (DCC)1 in patients with CF, whereas no isolates were related to DCC2; isolates related to DCC3 were not necessarily predominant in our sample set. We found that the erm(41) T28C mutants spread globally, and some of them reacquired the functional erm(41) gene through both point mutation and recombination. This study revealed predominant MABC clones in Japan and Taiwan and their relationship with the globally superadding clones in the patient community with CF. Our study provides insights into the genetic characteristics of globally dominant and area-specific strains isolated from patients with or without CF and differences between globally spread and regionally specific strains.IMPORTANCE Members of Mycobacterium abscessus complex (MABC) are frequently isolated from patients. Studies have reported that predominant clones of MABC (known as dominant circulating clones; DCCs) are distributed worldwide and transmitted from humans to humans in patients with cystic fibrosis (CF). However, associated genomic epidemiology has not yet been conducted in East Asia, including Japan and Taiwan, where there are only a few patients with CF. Using whole-genome sequencing data derived from non-CF patients in Japan and Taiwan, we revealed prevalent clones and the incidence of macrolide resistance-associated mutations in the MABC population in this region. We also clarified the associations between these predominant clones and DCCs in the global CF patient community. Our results would assist further studies in elucidating the genetic characteristics of strains isolated from patients with or without CF, the differences between globally spread and regionally specific strains, and the adaptive evolution of MABC within the host.