Clinical characteristics and logistic regression analysis of macrolide-resistant Mycoplasma pneumoniae pneumonia in children.

PURPOSE: To investigate macrolide-resistant Mycobacterium pneumoniae (MRMP) pneumonia in children and construct a logistic regression model for mutations in the Mycoplasma pneumoniae drug-resistant gene. METHODS: Clinical data of 281 children were analyzed. Sequencing confirmed a mutation at the A2063G locus of the 23 S rRNA gene in 227 children (A2063G group); 54 children showed no mutations (non-MRMP [NMRMP] group). We compared clinical features, laboratory tests, imaging, and bronchoscopy results and constructed a multifactorial logistic regression model to analyze risk and protective factors. RESULTS: The A2063G group had longer durations of fever and hospitalization before admission, a higher proportion of treatment with sodium methylprednisolone succinate (MPS)/dexamethasone, longer time to discontinue hormones, and higher probability of combined infections. Monocyte percentage was significantly higher in the A2063G group. Imaging suggested a higher incidence of infections in the right lung compared to both lungs. Univariate analysis revealed fever duration before admission, hormone dose and duration, monocyte percentage, and mixed infections as risk factors for Mycoplasma pneumoniae infection with the A2063G mutation. The logistic regression model showed that mixed infections were an independent risk factor for the A2063G locus mutation, whereas hormone dose was a protective factor. CONCLUSION: A prevalence of macrolide resistance of 80.8% among children was observed in the region. Logistic regression analysis revealed that co-infection with other respiratory pathogens is an independent risk factor for the development of resistance genes, while the use of hormone dosage acts as a protective factor.

Design, synthesis and structure-activity relationships of novel non-ketolides: 9-Oxime clarithromycin featured with seven-to thirteen-atom-length diamine linkers at 3-OH.

We report here on the structure-activity relationships of hybrids combining 3-descladinosyl clarithromycin with quinolones linked by extended diamine connectors. Several hybrids, exemplified by 23Bc, 23Be, 23Bf, 26Be, and 30Bc, not only restored potency against inducibly resistant pathogens but also exhibited significantly enhanced activities against constitutively resistant strains of Staphylococcus pneumoniae and Staphylococcus pyogenes, which express high-level resistance independent of clarithromycin or erythromycin induction. Additionally, the novel hybrids showed susceptibility against Gram-negative Haemophilus influenzae. Notably, hybrid 23Be demonstrated dual modes of action by inhibiting both protein synthesis and DNA replication in vitro and in vivo. Given these promising characteristics, 23Be emerges as a potential candidate for the treatment of community-acquired bacterial pneumonia.

Three-birds-with-one-stone: An eco-friendly and renewable humic acid-derived material application strategy for macrolide antibiotic detection and multifunctional composite film preparation.

This research proposes a simple and novel strategy for the green detection of antibiotics along with the reduction of microplastic and humic acid (HA) hazards. The entire process is based on a single-step solvent-sieving method to separate HA into insoluble (IHA) and soluble (SHA) components, subsequently recombining and designing the application according to the original characteristics of selected fractions in accordance with the zero-waste principle. IHA was applied as a dispersive solid phase extraction (DSPE) sorbent without chemical modification for the enrichment of trace MACs in complex biological matrices. The recovery of MACs was 74.06-100.84 % in the range of 2.5-1000 µg∙kg-1. Furthermore, SHA could be combined with biodegradable polyvinyl alcohol (PVA) to prepare multifunctional composite films. SHA endows the PVA film with favorable mechanical properties, excellent UV shielding as well as oxidation resistance performance. Compared with pure PVA, the tensile strength, toughness, antioxidant and UV-protection properties were increased to 157.3 Mpa, 258.6 MJ·m-3, 78.6 % and 60 % respectively. This study achieved a green and economically valuable utilization of all components of waste HA, introduced a novel approach for monitoring and controlling harmful substances and reducing white pollution. This has significant implications for promoting sustainable development and recovering valuable resources.

Current Mycoplasma pneumoniae outbreak: Don't forget its role in Bickerstaff encephalitis.

Mycoplasma pneumoniae is a common cause of community-acquired pneumonia in children and young adults. It is responsible of a broad array of extrapulmonary manifestations, the most severe affecting the central nervous system. We report a challenging diagnosis of macrolide-resistant M. pneumoniae-induced Bickerstaff encephalitis in a 16-year-old man.

A novel photoreduction deposition induced AuNPs/COFs composite for SERS detection of macrolide antibiotics.

As we all know, SERS (Surface-enhanced Raman spectroscopy) is widely used in sensing, analysis and detection. The covalent organic frameworks (COFs) have performed well as a material for supporting metal nanoparticles and facilitating analyte adsorption in SERS, which may greatly enhance the detection sensitivity and reproducibility. The synthesis of traditional metal/COFs composites involved chemical reduction methods, however, the resulting metallic NPs exhibited reduced capacity to enhance SERS due to their small particle sizes (usually <20 nm). This paper presented a novel photoreduction method for the facile growth of AuNPs (diameters: 75 nm) on COFs matrix under light control, which represents the first report of such synthesis on COF. Subsequently, the photoreduction deposition induced AuNPs/COFs composites, which served as highly sensitive and reproducible SERS-active substrates for capturing the spectral information of four types of macrolide antibiotics. The detection limits for the four macrolide antibiotics were determined to be 3.30 × 10-11, 3.43 × 10-10, 1.10 × 10-10 and 5.78 × 10-11 M, respectively, exhibiting excellent linear relationships within the concentration range of 10-10 to 10-3 M. Therefore, our proposed SERS method opens up a new idea for the development of SERS substrates and environmental safety monitoring, and it has great potential for ensuring food safety in the future.

Multi-Locus sequence typing of Treponema pallidum in male patients with genital ulcers in a public STD clinic in South Brazil: a new allele and almost complete macrolide resistance / Tipificación de Treponema pallidum mediante MLST en pacientes masculinos con úlceras genitales en una clínica pública de ETS en el sur de Brasil: un nuevo alelo y resistencia casi completa a los macrólidos. / Tipagem do Treponema pallidum por MLST em pacientes do sexo masculino com úlceras genitais em uma clínica pública de DST no Sul do Brasil: um novo alelo e resistência quase completa aos macrolídeos

Objective: Considering the lack of data on T. pallidum genotyping in Brazil, we aimed to study its strains and their resistance to macrolides in genital ulcers suggestive of syphilis.Methods: Men with genital ulcers suggestive of syphilis were invited to participate. Samples were collected with a dry cotton swab and immersed in a 0.9% NaCl solution. Detection was done by PCR amplification of 260 bp of the tpp47 gene. The PCR product was analyzed by electrophoresis in a 2% agarose gel containing 0.05% ethidium bromide. Positive PCR samples were analyzed by MLST (sequencing of chromosomal loci TP0136, TP0548, and TP0705). The A2058G and A2059G mutations in the 23S rRNA gene were evaluated by nested PCR. DNA sequencing was analyzed using Bioedit software (Tom Hall, USA). Genotyping was performed using the PubMLST online platform (Grillová scheme). Results: All subjects were residents of Porto Alegre and aged between 19 and 66 years. Of the 43 samples, 32 were positive for PCR for T. pallidum. Thirty strains were available for genotyping and belonged to the SS14-like (73.3%) or Nicholslike (20%) Clonal Complex. Three complete MLST profiles were identified (1.3.1; 9.7.3 and 28.7.3), and a new allele was identified in one sample (approved by pubMLST curators as TP0705-22). Only one sample did not present the 2058 mutation in the 23S rRNA gene.Conclusion: Our study identified genetic diversity in T. pallidum DNA using MLST with allelic variants for TP0136, TP0548, and TP0705, including a new allele. A single sample was characterized as genotypically susceptible to macrolides. All other samples (more than 95%) presented the A2058G mutation in the 23S rRNA gene, which causes resistance to macrolides. Improving understanding of the local epidemiology of T. pallidum with representative samples that allow cofactor analysis is crucial for prevention and care.

Objetivo: Considerando la falta de datos sobre el genotipado de T. pallidum en Brasil, nos propusimos estudiar sus cepas y su resistencia a macrólidos en úlceras genitales sugestivas de sífilis. Métodos: Se invitó a participar a hombres con úlceras genitales sugestivas de sífilis. Las muestras se recogieron con un hisopo de algodón seco y se sumergieron en una solución de NaCl al 0,9%. La detección se realizó mediante amplificación por PCR de 260 pb del gen tpp47. El producto de la PCR se analizó mediante electroforesis en un gel de agarosa al 2% que contenía bromuro de etidio al 0,05%. Las muestras de PCR positivas se analizaron mediante MLST (secuenciación de los loci cromosómicos TP0136, TP0548 y TP0705). Las mutaciones A2058G y A2059G en el gen 23S rRNA se evaluaron mediante PCR anidada. La secuenciación de ADN se analizó utilizando el software Bioedit (Tom Hall, EE. UU.). El genotipado se realizó utilizando la plataforma en línea PubMLST (esquema Grillová). Resultados: Todos los sujetos eran residentes de Porto Alegre y tenían edades comprendidas entre 19 y 66 años. De las 43 muestras, 32 resultaron positivas a la PCR para T. pallidum. Treinta cepas estaban disponibles para el genotipado y pertenecían al Complejo Clonal tipo SS14 (73,3%) o tipo Nichols (20%). Se identificaron tres perfiles MLST completos (1.3.1; 9.7.3 y 28.7.3) y se identificó un nuevo alelo en una muestra (aprobado por los curadores de pubMLST como TP0705-22). Sólo una muestra no presentó la mutación 2058 en el gen 23S rRNA. Conclusión: Nuestro estudio identificó diversidad genética en el ADN de T. pallidum utilizando MLST con variantes alélicas para TP0136, TP0548 y TP0705, incluido un nuevo alelo. Una sola muestra se caracterizó como genotípicamente susceptible a macrólidos. El resto de muestras (más del 95%) presentaron la mutación A2058G en el gen 23S rRNA, que provoca resistencia a los macrólidos. Mejorar la comprensión de la epidemiología local de T. pallidum con muestras representativas que permitan el análisis de cofactores es crucial para la prevención y la atención.

Objetivo: Considerando a ausência de dados sobre genotipagem de T. pallidum no Brasil, objetivamos estudar suas cepas e sua resistência aos macrolídeos em úlceras genitais sugestivas de sífilis.Métodos: Foram convidados a participar homens com úlceras genitais sugestivas de sífilis. As amostras foram coletadas com swab de algodão seco e imersas em solução de NaCl 0,9%. A detecção foi feita por amplificação por PCR de 260 pb do gene tpp47. O produto de PCR foi analisado por eletroforese em gel de agarose a 2% contendo brometo de etídio a 0,05%. Amostras de PCR positivas foram analisadas por MLST (sequenciamento dos loci cromossômicos TP0136, TP0548 e TP0705). As mutações A2058G e A2059G no gene 23S rRNA foram avaliadas por nested PCR. O sequenciamento de DNA foi analisado utilizando o software Bioedit (Tom Hall, EUA). A genotipagem foi realizada utilizando a plataforma online PubMLST (esquema Grillová).Resultados: Todos os sujeitos eram residentes de Porto Alegre e tinham idade entre 19 e 66 anos. Das 43 amostras, 32 foram positivas para PCR para T. pallidum. Trinta cepas estavam disponíveis para genotipagem e pertenciam ao Complexo Clonal SS14-like (73,3%) ou Nichols-like (20%). Três perfis completos de MLST foram identificados (1.3.1; 9.7.3 e 28.7.3), e um novo alelo foi identificado em uma amostra (aprovado pelos curadores do pubMLST como TP0705-22). Apenas uma amostra não apresentava a mutação 2058 no gene 23S rRNA.Conclusão: Nosso estudo identificou diversidade genética no DNA de T. pallidum usando MLST com variantes alélicas para TP0136, TP0548 e TP0705, incluindo um novo alelo. Uma única amostra foi caracterizada como genotípicamente suscetível a macrolídeos. Todas as demais amostras (mais de 95%) apresentaram a mutação A2058G no gene 23S rRNA, que causa resistência aos macrolídeos. Melhorar a compreensão da epidemiologia local do T. pallidum com amostragens representativas que permitam análise de cofatores é crucial para a prevenção e o cuidado.

Aberrant spliceosome activity via elevated intron retention and upregulation and phosphorylation of SF3B1 in chronic lymphocytic leukemia.

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

Effects of quorum sensing-interfering agents, including macrolides and furanone C-30, and an efflux pump inhibitor on nitrosative stress sensitivity in Pseudomonas aeruginosa.

Long-term administration of certain macrolides is efficacious in patients with persistent pulmonary Pseudomonas aeruginosa infection, despite how limited the clinically achievable concentrations are, being far below their MICs. An increase in the sub-MIC of macrolide exposure-dependent sensitivity to nitrosative stress is a typical characteristic of P. aeruginosa. However, a few P. aeruginosa clinical isolates do not respond to sub-MIC of macrolide treatment. Therefore, we examined the effects of sub-MIC of erythromycin (EM) on the sensitivity to nitrosative stress together with an efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN). The sensitivity to nitrosative stress increased, suggesting that the efflux pump was involved in inhibiting the sub-MIC of macrolide effect. Analysis using efflux pump-mutant P. aeruginosa revealed that MexAB-OprM, MexXY-OprM, and MexCD-OprJ are factors in reducing the sub-MIC of macrolide effect. Since macrolides interfere with quorum sensing (QS), we demonstrated that the QS-interfering agent furanone C-30 (C-30) producing greater sensitivity to nitric oxide (NO) stress than EM. The effect of C-30 was decreased by overproduction of MexAB-OprM. To investigate whether the increase in the QS-interfering agent exposure-dependent sensitivity to nitrosative stress is characteristic of P. aeruginosa clinical isolates, we examined the viability of P. aeruginosa treated with NO. Although treatment with EM could reduce cell viability, a high variability in EM effects was observed. Conversely, C-30 was highly effective at reducing cell viability. Treatment with both C-30 and PAßN was sufficiently effective against the remaining isolates. Therefore, the combination of a QS-interfering agent and an EPI could be effective in treating P. aeruginosa infections.

Mycoplasma genitalium prevalence, co-infection and macrolide resistance-associated mutations in Southern Vietnam.

Mycoplasma genitalium is an emerging sexually transmitted infection, with increasing rates of macrolide resistance and some ways of treatments being recommended by many countries. This study aimed to investigate the prevalence of M. genitalium infection, M. genitalium co-infection with other sexually transmitted organisms, and the frequency of macrolide antibiotic resistance genotypes identified in urethral specimens collected from male and urethral, vaginal and cervical specimens from female who visited the STIs clinic of HCMC Hospital of Dermato-Venereology, Vietnam. The results obtained positive samples for C. trachomatis was 8.46%, N. gonorrhoeae was 6.28%, and M. genitalium was 5.95%. Fifty-five out of 90 M. genitalium samples were found to have mutations in the 23S rRNA gene associated with macrolide resistance (61.11%). M. genitalium/C. trachomatis co-infection was 6.19%, and M. genitalium/N. gonorrhoeae was 1.22%. The percentage of M. genitalium carrying the macrolide resistance mutant gene co-infected with C. trachomatis accounted for 37.50%. The high prevalence of the M. genitalium mutations associated with macrolide resistance showed the importance of M. genitalium testing.

Necrotizing pneumonia associated with macrolide-resistant Mycoplasma in a child.

We reported a pediatric case of necrotizing pneumonia due to macrolide-resistant Mycoplasma pneumoniae, an uncommon presentation of a common disease. Acquisition of resistance does not increase virulence, but it leads to more difficult treatment and potential complications. Macrolide-resistant M. pneumoniae requires extended antibiotic therapy with the addition of a second-line agent and an immunomodulator to promote clinical improvement with minimal sequelae.

Iatrogenically Acquired Mycobacterium abscessus Infection in an Indwelling Intercostal Drainage In Situ in a Patient With Alcoholic Liver Disease and Bilateral Hepatic Hydrothorax: A Report of a Rare Case.

A 47-year-old male, a known case of alcoholic chronic liver disease with portal hypertension, presented with complaints of abdominal distension and shortness of breath. A provisional diagnosis of ethanol-related compensated chronic liver disease (CLD) with portal hypertension and splenomegaly, gross ascites with bilateral hepatic hydrothorax was made. The left-sided pleural effusion subsided after three pleural taps, but the right-sided effusion kept refilling even after four to five days of repeated therapeutic taps, so a pigtail catheter was left in situ. The pleural fluid was sent for culture which did not grow any pathogenic organisms. Cartridge-based nucleic acid amplification tests where Mycobacterium tuberculosis complex (MTBC) was not detected, Ziehl-Neelsen staining was done in which acid-fast bacilli were not seen, and cytology was done where no malignant cells were seen. The patient was discharged with the pigtail in situ on the right side and, after 20 days, the patient again presented with shortness of breath, and imaging revealed moderate right-side pleural effusion. Draining of pleural fluid was done and sent for investigation which again revealed no infective etiology. The patient was admitted to the hospital for one month as the right-sided effusion did not resolve. Suddenly, the patient developed shortness of breath, and a chest X-ray was done, which showed pigtail blockage; pigtail flushing was done, and the bag was drained. The patient was empirically started on IV meropenem 500 mg TID, IV teicoplanin 400 mg BD, and inj polymyxin B 500,000 IU IV BD. The pleural fluid was sent continuously for investigation for the first two months which again did not reveal any infective etiology. After two months of pigtail in situ, the pleural fluid was sent for CBNAAT where MTBC was not detected, and ZN stain showed smooth acid-fast bacilli. The sample was cultured, and it grew acid-fast bacilli in 72 hours on blood agar, MacConkey agar, and Lowenstein-Jensen media. A line probe assay done from the isolate revealed it to be Mycobacterium abscessus subsp. abscessus which was resistant to macrolides and sensitive to aminoglycosides. Mycobacterium abscessus subsp. abscessus was isolated from repeated cultures of pleural fluid, and the patient was advised on a combination treatment of amikacin, tigecycline, and imipenem. The patient was discharged with the indwelling pigtail with the advised treatment; unfortunately, we lost patient follow-up as the patient never returned to us.

Total Synthesis of an Epothilone Analogue based on the Amide‒Triazole Bioisosterism.

Epothilones are 16-membered macrolides that can act as microtubule-targeting agents to tackle cancer. Many synthetic analogues have been investigated for their activity, yet often based on macrolide structures. A notable exception is Ixabepilone, an azalide representing the only epothilone-like molecule approved by the FDA as a chemotherapeutic. Exploiting the amide-triazole bioisosterism, in this work we report the synthesis of the first generation of epothilones lacking the macrolide or azalide structure, with the ester or amide linkage replaced by a triazole unit. Together with the synthesis of this new analogue, computational and biological evaluations have been performed too.

Xylatolides A and B, new 10-membered macrolides from the endophytic fungus Xylaria sp.

Chemical investigation of the fungal endophyte Xylaria sp. isolated from leaves of Moringa oleifera, collected in Cameroon, resulted in the previously undescribed 10-membered macrolide, and two known natural products. The structures of the xylatolides A and B were unambiguously identified by their mass spectra and by extensive 1D and 2D NMR spectroscopic analysis, featuring a 10-membered lactone core structure with oxygenated substituents and an unsubstituted 10-alkyl chain presenting seven carbon atoms. Compounds were screened for their cytotoxic potential against the human HepG2 hepatocellular carcinoma cells and HCT-116 cells (human colon carcinoma cell line). Moreover, the isolated compounds were also assayed against a small panel of sensitive strains including the bacterial species Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis as well as against the fungal species Candida albicans. However, no significant activities were found.

Formulation and Stability of a 1% Clarithromycin-Based Topical Skin Cream: A New Option to Treat Buruli Ulcers?

There are more than 170 known species of non-tuberculous mycobacteria, and some are responsible for serious diseases in people infected with them. One of these is Buruli ulcers, a neglected tropical disease endemic in more than 33 countries and caused by Mycobacterium ulcerans, which infects skin tissue. Treatment consists of a long-term regimen combining the use of oral rifampin with another anti-tuberculosis drug (e.g., clarithromycin). Patients in these countries face difficulties in accessing and adhering to this therapy. This study investigates the feasibility of formulating stable, optimized clarithromycin as a topical cutaneous cream. The cream was formulated, and its stability was evaluated under different storage temperature conditions and using a stability indicator method. The results showed that the clarithromycin cream was stable for at least 60 days, even at extreme temperatures (40 °C). In conclusion, the data presented here demonstrate the stability of a new form of topical cutaneous clarithromycin, which may offer a new approach to the treatment of Buruli ulcers and clarithromycin-sensitive infections.

The intrinsic macrolide resistome of Escherichia coli.

Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and molecular mechanisms remain to be fully elucidated. Here, we used transposon directed insertion-site sequencing (TraDIS) to identify chromosomal non-essential genes involved in Escherichia coli intrinsic resistance to a macrolide antibiotic, tilmicosin. We constructed two highly saturated transposon mutant libraries of >290,000 and >390,000 unique Tn5 insertions in a clinical enterotoxigenic strain (ETEC5621) and in a laboratory strain (K-12 MG1655), respectively. TraDIS analysis identified genes required for growth of ETEC5621 and MG1655 under 1/8 MIC (n = 15 and 16, respectively) and 1/4 MIC (n = 38 and 32, respectively) of tilmicosin. For both strains, 23 genes related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump, and peptidoglycan metabolism were enriched in the presence of the antibiotic. Individual deletion of genes (n = 10) in the wild-type strains led to a 64- to 2-fold reduction in MICs of tilmicosin, erythromycin, and azithromycin, validating the results of the TraDIS analysis. Notably, deletion of surA or waaG, which impairs the outer membrane, led to the most significant decreases in MICs of all three macrolides in ETEC5621. Our findings contribute to a genome-wide understanding of intrinsic macrolide resistance in E. coli, shedding new light on the potential role of the peptidoglycan layer. They also provide an in vitro proof of concept that E. coli can be sensitized to macrolides by targeting proteins maintaining the outer membrane such as SurA and WaaG.

Comparative genomic sequencing to characterize Mycoplasma pneumoniae genome, typing, and drug resistance.

To analyze the characteristics of Mycoplasma pneumoniae as well as macrolide antibiotic resistance through whole-genome sequencing and comparative genomics. Thirteen clinical strains isolated from 2003 to 2019 were selected, 10 of which were resistant to erythromycin (MIC >64 µg/mL), including 8 P1-type I and 2 P1-type II. Three were sensitive (<1 µg/mL) and P1-type II. One resistant strain had an A→G point mutation at position 2064 in region V of the 23S rRNA, the others had it at position 2063, while the three sensitive strains had no mutation here. Genome assembly and comparative genome analysis revealed a high level of genome consistency within the P1 type, and the primary differences in genome sequences concentrated in the region encoding the P1 protein. In P1-type II strains, three specific gene mutations were identified: C162A and A430G in L4 gene and T1112G mutation in the CARDS gene. Clinical information showed seven cases were diagnosed with severe pneumonia, all of which were infected with drug-resistant strains. Notably, BS610A4 and CYM219A1 exhibited a gene multi-copy phenomenon and shared a conserved functional domain with the DUF31 protein family. Clinically, the patients had severe refractory pneumonia, with pleural effusion, necessitating treatment with glucocorticoids and bronchoalveolar lavage. The primary variations between strains occur among different P1-types, while there is a high level of genomic consistency within P1-types. Three mutation loci associated with specific types were identified, and no specific genetic alterations directly related to clinical presentation were observed.IMPORTANCEMycoplasma pneumoniae is an important pathogen of community-acquired pneumonia, and macrolide resistance brings difficulties to clinical treatment. We analyzed the characteristics of M. pneumoniae as well as macrolide antibiotic resistance through whole-genome sequencing and comparative genomics. The work addressed primary variations between strains that occur among different P1-types, while there is a high level of genomic consistency within P1-types. In P1-type II strains, three specific gene mutations were identified: C162A and A430G in L4 gene and T1112G mutation in the CARDS gene. All the strains isolated from severe pneumonia cases were drug-resistant, two of which exhibited a gene multi-copy phenomenon, sharing a conserved functional domain with the DUF31 protein family. Three mutation loci associated with specific types were identified, and no specific genetic alterations directly related to clinical presentation were observed.

Macrolide and fluoroquinolone resistance associated mutations in Mycoplasma genitalium in men who have sex with men attending STI clinic: A pilot study from India.

Background Increasing rates of macrolide and fluroquinolone resistance in Mycoplasma genitalium (MG) are being reported worldwide with resultant treatment failure. Aims and objectives We aimed to determine the level of antibiotic resistance of MG in men who have sex with men (MSM) attending a sexually transmitted infections (STIs) clinic in New Delhi, India. Methods Real-time polymerase chain reaction (PCR) assays targeting MgPa and pdhD genes were performed to detect MG rectal, urogenital or oropharyngeal infections in 180 MSM between January 2022 and June 2023. Macrolide resistance-associated mutations (MRM) and quinolone resistance-associated mutations (QRM) were detected by specific amplification of domain V of 23SrRNA gene and appropriate regions of parC and gyrA genes respectively followed by sequencing. PCR-based screening for Chlamydia trachomatis (CT) infection was also performed. Results A total of 13 (7.2%) MSM were positive for MG infection. The most common site of infection was anorectum (8/13; 61.5%) followed by the urethra (5/13; 38.5%). None of the patients had infection at both the sites, and no oropharyngeal MG infection was detected. CT infection was detected in 37 (20.6%) MSM. Of the 13 MG-infected MSM, 6 (46.2%) were co-infected with CT. MRM and QRM were found in five (46.2%) and two (15.4%) strains, respectively. Both Quinolone resistance mutation (QRM)-harbouring strains also harboured MRM. All the five MG isolates carried the MRM A2071G. Both the QRM isolates co-harboured the parC and gyrA single-nucleotide polymorphisms. There was no correlation between the presence of antibiotic resistance and co-infection with CT (P = 0.52). Limitation Because all patients in the study were MSM, the high rate of resistance to macrolides and fluoroquinolones could not be extrapolated for non-MSM patients. Conclusion This is a report of an initial survey of antibiotic resistance to MG in a country where its diagnosis and treatment are not routinely available. We found a high prevalence of MG-carrying MRM, QRM and dual-class resistance in MSM in the absence of antibiotic exposure. This study mandates the need for both screening and detection of antimicrobial resistance against MG.

Mycoplasmoides genitalium nucleic acid semi-quantitation and molecular macrolide resistance detection via automated assays: gender and specimen source considerations.

A laboratory-developed test (LDT) using analyte-specific reagents has been optimized on a commercial platform to detect macrolide resistance-associated mutations (MRM) in 23S rRNA from Mycoplasmoides genitalium from primary clinical specimens. In this study, MRM-LDT was applied to a multi-specimen source study set. One thousand four hundred ninety-five primary specimens testing positive for M. genitalium by commercial transcription-mediated amplification (TMA) were initially titered by the TMA assay using serial 10-fold dilutions to semi-quantitate target nucleic acid burden. Primary specimens were then processed for MRM detection using the MRM-LDT. Findings were stratified by gender and specimen source. The mean log10 target nucleic acid titer of a TMA-positive specimen was 3.51 (median 3; range 0-10). Male specimens (n = 1145) demonstrated a mean log10 M. genitalium TMA titer of 3.67; that value observed in 350 female specimens was 2.98 (P < 0.0001). The MRM-LDT detection rate (88.7%) from specimens with log10 M. genitalium TMA titers ≥ 4 was increased over specimens with log10 titers ≤ 1 (4.5%; P < 0.0002). In females, MRM-LDT was positive from 51.3% of vaginal swab and 34.7% of urine specimens (P = 0.01). In males, MRM-LDT was positive from 65.0% of rectal swab and 55.7% of urine specimens (P = 0.002). Differences were also observed in log10 M. genitalium TMA titers as a function of specimen source. M. genitalium macrolide resistance rates among multiple specimen sources, as determined by MRM-LDT, are high in the United States and can be consistent with target nucleic acid burden within the primary specimen. Caveats experienced within subgroupings support MRM reflex testing on primary M. genitalium-positive specimens. IMPORTANCE: First-line macrolide treatment failure is of increasing concern with Mycoplasmoides genitalium in multiple settings. Recent sexually-transmitted infection treatment guidelines from the United States Centers for Disease Control and Prevention have predicated therapeutic approaches on the availability of a macrolide resistance/susceptibility result from a primary clinical specimen. In this report, we investigate potential correlation between macrolide resistance mutation detection rates (identified by a molecular amplified laboratory-developed test) and transcription-mediated amplification-based rRNA target semi-quantitation. Data reveal that rRNA semi-quantitation and laboratory-developed test detection rate differences exist as a function of gender and specimen source. These data can guide providers in proper specimen selection not only for the laboratory diagnosis of M. genitalium but also macrolide resistance mutation determination from primary clinical specimens.

Exploring the Molecular Mechanisms of Macrolide Resistance in Laboratory Mutant Helicobacter pylori.

Resistance to clarithromycin, a macrolide antibiotic used in the first-line treatment of Helicobacter pylori infection, is the most important cause of treatment failure. Although most cases of clarithromycin resistance in H. pylori are associated with point mutations in 23S ribosomal RNA (rRNA), the relationships of other mutations with resistance remain unclear. We examined possible new macrolide resistance mechanisms in resistant strains using next-generation sequencing. Two resistant strains were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations using the agar dilution method. Sanger sequencing and whole-genome sequencing were performed to detect resistance-related mutations. Both strains carried the A2142G mutation in 23S rRNA. Candidate mutations (T1495A, T1494A, T1490A, T1476A, and G1472T) for clarithromycin resistance were detected in the Mutant-1 strain. Furthermore, a novel mutation in the gene encoding for the sulfite exporter TauE/SafE family protein was considered to be linked to clarithromycin resistance or cross-resistance, being identified as a target for further investigations. In the Mutant-2 strain, a novel mutation in the gene that encodes DUF874 family protein that can be considered as relevant with antibiotic resistance was detected. These mutations were revealed in the H. pylori genome for the first time, emphasizing their potential as targets for advanced studies.