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.

Accurate identification of non-tuberculous mycobacterial (NTM) species is crucial for the diagnosis and appropriate management of NTM infections. This study aimed to evaluate the performance of two assays, FluoroType Mycobacteria VER 1.0 and Maldi BioTyper (MBT) Mycobacteria. The two assays were evaluated using 119 NTM, including 85 slow-growing mycobacteria and 34 rapid-growing mycobacteria, representing a total of 33 species isolated in three French clinical laboratories. We used the GenoType assays as reference method for species identification, followed by 16S rRNA gene sequencing if the GenoType kits returned Mycobacterium sp. Compared to the reference method, the FluoroType Mycobacteria assay provided correct species identification in 89.9% of cases (107/119). Among the most frequently encountered species in clinical settings, low concordance was obtained for Mycobacterium intracellulare (82.4%, 14/17), Mycobacterium gordonae (66.7%, 6/9), and Mycobacterium xenopi (75%, 6/8). Misidentification was obtained in two cases ( Mycobacterium smegmatis instead of Mycobacterium mageritense , and Mycobacterium mucogenicum instead of Mycobacterium phocaicum ). Using the MBT Mycobacteria assay, 78.1% (93/119) of NTM isolates were correctly identified at the species level. One Mycobacterium europaeum isolate was misidentified as M. intracellulare / Mycobacterium chimaera . In five cases, the assay provided more accurate NTM identification compared to GenoType assays, in which closely related species are identified as a group. The FluoroType Mycobacteria VER 1.0 and the MBT Mycobacteria assays are useful tools for NTM identification from positive cultures, reducing handling time compared to GenoType assays. Their routine use in laboratories must take into consideration their performance and limitations in clinical settings.

Unprecedented global increases in Mycoplasma pneumoniae respiratory infections began in late summer 2023. Although a predominance of p1 subtype 1 associated with alarmingly high rates of macrolide resistance was reported in Southeast Asia, data from Europe remain limited. We aimed to characterise the circulating M. pneumoniae strains in French hospitalised patients, using a high-throughput amplicon sequencing approach. Phylogenetic analysis was performed using p1 adhesin gene subtyping and multi-locus sequence typing. Mutations associated with antibiotic resistance were screened in the 23S and 16S rRNA, rplD, rplV, gyrA, and parC genes. Although most patients (835/933; 89.5%) presented with respiratory symptoms, extrapulmonary manifestations were observed in 49/884 (5.5%) patients. Five p1 subtypes and 29 distinct sequence types were identified, with a predominance of p1 subtype 2 (67.2%) and ST14 (29.7%). Subtype 2 was more frequent in adults, whereas subtype 1 was more often associated with extrapulmonary manifestations. Macrolide resistance-associated mutations were observed at a 3.0% prevalence, exhibiting a trend toward subtype 1 and high frequency of mixed populations. No mutations clearly associated with fluoroquinolone or tetracycline resistance were identified. This study demonstrates the polyclonal spread of M. pneumoniae strains and a low prevalence of macrolide resistance during the 2023-2024 outbreak in France.

Macrolides are the first-line treatment for Mycoplasma pneumoniae infections; however, macrolide resistance has been reported. We evaluated the clinical performance of two commercial assays for detecting macrolide-resistant M. pneumoniae : the LightMix Modular Mycoplasma Macrolide (TIB Molbiol) and the Mycoplasma Pneumoniae and Macrolides-Resistant Strain Nucleic Acid Test Kit (Mole Bioscience), using 23S rRNA Sanger sequencing as the reference method. Eight M. pneumoniae strains, 10 non- M . pneumoniae Mycoplasma strains, and 237 clinical samples were tested. Overall, the Mole Bioscience kit failed to detect M. pneumoniae in 27.7% of positive samples, whereas the TIB Molbiol kit missed only 5.1%. The clinical sensitivities for detecting macrolide resistance–associated mutations in clinical samples were 90.9% for the TIB Molbiol kit and 81.5% for the Mole Bioscience kit. All false wild-type results corresponded to samples harboring a mutation at position 2067 ( M. pneumoniae numbering) of the 23S rRNA gene. The clinical specificities were 97.9% and 94.4% for the TIB Molbiol and Mole Bioscience kits, respectively. Notably, the TIB Molbiol kit also detected 23S rRNA mutations in Mycoplasma genitalium and Mycoplasma amphoriforme , whereas the Mole Bioscience kit yielded false-resistant results in high-load specimens. In conclusion, these commercial kits offer a convenient approach for detecting macrolide resistance in routine clinical practice. The Mole Bioscience kit demonstrated limited sensitivity for M. pneumoniae detection, restricting its utility for resistance identification, and neither assay reliably detected resistance-associated mutations at position 2067. The global M. pneumoniae reemergence highlights the need for advanced assays detecting all macrolide resistance-associated mutations. IMPORTANCE: Mycoplasma pneumoniae is a leading cause of bacterial respiratory tract infections, and a marked resurgence was reported worldwide in autumn 2023. Macrolides are the recommended first-line treatment for M. pneumoniae infections; however, resistance rates vary widely across regions and may compromise therapeutic efficacy. Rapid identification of macrolide resistance is therefore critical to ensure appropriate patient management. Because culture-based methods are slow and have limited sensitivity, molecular assays have become central to both pathogen detection and resistance determination. However, few simple and reliable commercial assays have been developed and evaluated for the detection of macrolide resistance in routine diagnostic laboratories. In this study, we evaluated the clinical performance of two real-time PCR–based commercial assays for detecting macrolide resistance–associated mutations in M. pneumoniae . Our findings support the implementation of rapid molecular tools to guide timely therapeutic decisions, including the early use of second-line antimicrobials when resistance is detected.

Direct genome sequencing of Mycoplasma genitalium from clinical specimens is challenging due to the organism’s low bacterial load. We developed and compared two DNA enrichment strategies—amplicon-based and hybridisation capture-based—coupled with next-generation sequencing, and assessed the suitability of DNA capture for whole-genome sequencing and high-resolution molecular typing. The enrichment approaches, RNA bait hybridisation and targeted sequence amplification, were combined with paired-end sequencing on the Illumina iSeq 100. Method performance was evaluated in 89 M. genitalium -positive specimens across five genomic loci: 23S and 16S rRNA, and parC and gyrA for macrolide, tetracycline, and fluoroquinolone resistance, respectively, and mgpB for phylogenetic analysis. Regarding antimicrobial resistance (AMR) determinants, the multiplex amplicon sequencing method demonstrated the highest sensitivity, at 93.3–98.9%. Although < 50% of samples were characterised using DNA capture, concordance between the two methods was excellent. Using DNA capture, 24.7% of specimens achieved a minimum coverage of 95% at 1× depth. Sequencing success was inversely correlated with human DNA contamination and the presence of low-quality reads. Whole-genome single-nucleotide polymorphism analysis provided higher discriminatory power than multi-locus sequence typing schemes and confirmed the clonality of multidrug-resistant M. genitalium strains belonging to the mgpB genotype 159. In conclusion, targeted amplicon-based enrichment is the most accurate and reliable approach for epidemiological studies focused on AMR and mgpB typing, whereas DNA capture is valuable for generating comprehensive genomic data from selected M. genitalium –positive specimens. Impact statement: Culturing Mycoplasma genitalium from clinical specimens is challenging; consequently, epidemiological studies rely on molecular techniques. Whole-genome sequences have been obtained from a few M. genitalium clinical isolates, and direct sequencing from clinical specimens requires enrichment strategies to overcome the organism’s low bacterial load. Consequently, published data on antimicrobial resistance (AMR) mechanisms and phylogenetic relationships among circulating strains remain incomplete. We demonstrated that targeted amplicon-based enrichment provided high-resolution AMR detection and highly sensitive mgpB genotyping, enabling the identification of minority variants. In contrast, hybridisation capture–based enrichment showed lower sensitivity but permitted successful WGS in a subset of M. genitalium –positive specimens and supported the development of new typing schemes. Although amplicon-based enrichment remains a reliable approach for epidemiological studies, strategic application of DNA capture facilitates the generation of comprehensive genomic data even though performance is reduced in the context of low bacterial loads, high host DNA contamination or suboptimal DNA quality.