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Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell.
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Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , ADN Bacteriano/genética , Genoma Bacteriano , Mycoplasma/genética , Biología Sintética/métodos , Proteínas Bacterianas/antagonistas & inhibidores , Sistemas CRISPR-Cas , Ingeniería GenéticaRESUMEN
Certain obligate parasites induce complex and substantial phenotypic changes in their hosts in ways that favor their transmission to other trophic levels. However, the mechanisms underlying these changes remain largely unknown. Here we demonstrate how SAP05 protein effectors from insect-vectored plant pathogenic phytoplasmas take control of several plant developmental processes. These effectors simultaneously prolong the host lifespan and induce witches' broom-like proliferations of leaf and sterile shoots, organs colonized by phytoplasmas and vectors. SAP05 acts by mediating the concurrent degradation of SPL and GATA developmental regulators via a process that relies on hijacking the plant ubiquitin receptor RPN10 independent of substrate ubiquitination. RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10. A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities. Therefore, one effector protein enables obligate parasitic phytoplasmas to induce a plethora of developmental phenotypes in their hosts.
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Arabidopsis/crecimiento & desarrollo , Arabidopsis/parasitología , Interacciones Huésped-Parásitos/fisiología , Parásitos/fisiología , Proteolisis , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Ingeniería Genética , Humanos , Insectos/fisiología , Modelos Biológicos , Fenotipo , Fotoperiodo , Filogenia , Phytoplasma/fisiología , Desarrollo de la Planta , Brotes de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Reproducción , Nicotiana , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The human microbiota affects critical cellular functions, although the responsible mechanism(s) is still poorly understood. In this regard, we previously showed that Mycoplasma fermentans DnaK, an HSP70 chaperone protein, hampers the activity of important cellular proteins responsible for DNA integrity. Here, we describe a novel DnaK knock-in mouse model generated in our laboratory to study the effect of M. fermentans DnaK expression in vivo. By using an array-based comparative genomic hybridization assay, we demonstrate that exposure to DnaK was associated with a higher number of DNA copy number variants (CNVs) indicative of unbalanced chromosomal alterations, together with reduced fertility and a high rate of fetal abnormalities. Consistent with their implication in genetic disorders, one of these CNVs caused a homozygous Grid2 deletion, resulting in an aberrant ataxic phenotype that recapitulates the extensive biallelic deletion in the Grid2 gene classified in humans as autosomal recessive spinocerebellar ataxia 18. Our data highlight a connection between components of the human urogenital tract microbiota, namely Mycoplasmas, and genetic abnormalities in the form of DNA CNVs, with obvious relevant medical, diagnostic, and therapeutic implications.
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Variaciones en el Número de Copia de ADN , Infecciones por Mycoplasma , Mycoplasma fermentans/genética , Homocigoto , Infecciones por Mycoplasma/genética , Infecciones por Mycoplasma/metabolismo , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
Kawasaki disease (KD) is a systemic vasculitis with an unknown cause that primarily affects children. The objective of this study was to explore the function and underlying mechanism of mitophagy in Mycoplasma pneumoniae (MP)-induced KD. To create MP-induced KD models, Human coronary endothelial cells (HCAECs) and DBA/2 mice were employed and treated with Mp-Lipid-associated membrane proteins ï¼LAMPsï¼. Lactate dehydrogenase (LDH) levels were tested to determine cellular damage or death. The inflammatory cytokines tumor necrosis factor (TNF)--α and interleukin (IL)-6 were measured using the Enzyme-Linked Immunosorbent Assay (ELISA) method. RT-qPCR and Western blotting were used to determine the expression of Intercellular Adhesion Molecule(ICAM)-1, vascular cell adhesion molecule (VCAM)-1, inducible nitric oxide synthase(iNOS), LC3, p62, PINK1(a mitochondrial serine/threonine-protein kinase), and PARKIN(a cytosolic E3-ubiquitin ligase). The adenosine triphosphate ï¼ATPï¼, reactive oxygen species ï¼ROSï¼, and mitochondrial membrane potentialï¼MMPï¼ levels were measured to determine mitochondrial function. Mitophagy was investigated using immunofluorescence and a mitophagy detection test. Autophagosome and mitochondrial morphology were examined using transmission electron microscopy. To identify inflammatory cell infiltration, hematoxylin and eosin staining was utilized. Mp-LAMPs increased the levels of TNF-α, IL-6, ICAM-1, VCAM-1, and iNOS in an HCAEC cell model, along with LDH release. After Mp-LAMPs exposure, there was a rise in LC3 and a reduction in p62. Meanwhile, the expression of PINK1 and Parkin was increased. Cyclosporin A dramatically increased ATP synthesis and MMP in HCAEC cells treated with Mp-LAMPs, while suppressing ROS generation, demonstrating excessive mitophagy-related mitochondrial dysfunction. Additionally, neither body weight nor artery tissue were affected due to PINK1 and Parkin suppression Cyclosporin A in Mp-LAMPs-treated mice. These findings indicated that PINK1/Parkin-mediated mitophagy inhibition may be a therapeutic target for MP-induced KD.
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Mitofagia , Síndrome Mucocutáneo Linfonodular , Mycoplasma pneumoniae , Proteínas Quinasas , Ubiquitina-Proteína Ligasas , Animales , Síndrome Mucocutáneo Linfonodular/metabolismo , Síndrome Mucocutáneo Linfonodular/patología , Proteínas Quinasas/metabolismo , Humanos , Ratones , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Mycoplasma pneumoniae/patogenicidad , Ratones Endogámicos DBA , Células Endoteliales/metabolismo , Células Endoteliales/patología , Neumonía por Mycoplasma/metabolismo , Neumonía por Mycoplasma/patología , Neumonía por Mycoplasma/microbiología , Mitocondrias/metabolismo , Mitocondrias/patología , Especies Reactivas de Oxígeno/metabolismo , Potencial de la Membrana MitocondrialRESUMEN
Mycoplasma hominis, a commensal bacterium that commonly inhabits the genital tract, leading to infections in both the genitourinary and extragenital regions. However, the antimicrobial resistance and pathogenic mechanisms of M. hominis isolated from extra-urogenital cystic abscess is largely unknown. This study reports the genomic epidemiological characteristics of a M. hominis isolate recovered from a pelvic abscess sample in China. Genomic DNA was extracted and sequenced using Illumina HiSeq X Ten platform. De novo assembly was performed and in silico analysis was accomplished by multiple bioinformatics tools. For phylogenomic analysis, publicly available M. hominis genomes were retrieved from NCBI GenBank database. Whole genome sequencing data showed that the genome size of M. hominis MH4246 was calculated as 679,746 bp, with 558 protein-coding sequences and a G + C content of 26.9%. M. hominis MH4246 is resistant to fluoroquinolones and macrolides, harboring mutations in the quinolone resistance-determining regions (QRDRs) (GyrA S153L, ParC S91I and ParE V417I) and 23S rRNA gene (G280A, C1500T, T1548C and T2218C). Multiple virulence determinants, such as tuf, hlyA, vaa, oppA, MHO_0730 and alr genes, were identified. Phylogenetic analysis showed that the closest relative of M. hominis MH4246 was the strain MH-1 recovered from China, which differed by 3490 SNPs. Overall, this study contributes to the comprehension of genomic characteristics, antimicrobial resistance patterns, and the mechanisms underlying the pathogenicity of this pathogen.
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Absceso , Mycoplasma hominis , Humanos , Mycoplasma hominis/genética , Filogenia , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Fluoroquinolonas/farmacología , Fluoroquinolonas/uso terapéuticoRESUMEN
Bacterial actin MreB forms filaments composed of antiparallel double-stranded units. The wall-less helical bacterium Spiroplasma has five MreB homologs (MreB1-5), some of which are involved in an intracellular ribbon for driving the bacterium's swimming motility. Although the interaction between MreB units is important for understanding Spiroplasma swimming, the interaction modes of each ribbon component are unclear. Here, we examined the assembly properties of Spiroplasma eriocheiris MreB5 (SpeMreB5), one of the ribbon component proteins that forms sheets. Electron microscopy revealed that sheet formation was inhibited under acidic conditions and bundle structures were formed under acidic and neutral conditions with low ionic strength. We also used solution assays and identified four properties of SpeMreB5 bundles as follows: (I) bundle formation followed sheet formation; (II) electrostatic interactions were required for bundle formation; (III) the positively charged and unstructured C-terminal region contributed to promoting lateral interactions for bundle formation; and (IV) bundle formation required Mg2+ at neutral pH but was inhibited by divalent cations under acidic pH conditions. During these studies, we also characterized two aggregation modes of SpeMreB5 with distinct responses to ATP. These properties will shed light on SpeMreB5 assembly dynamics at the molecular level.
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Actinas , Proteínas Bacterianas , Movimiento , Spiroplasma , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Cationes Bivalentes/metabolismo , Concentración de Iones de Hidrógeno , Magnesio/metabolismo , Movimiento/fisiología , Spiroplasma/fisiologíaRESUMEN
Enzootic pneumonia caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) has inflicted substantial economic losses on the global pig industry. The progression of M. hyopneumoniae induced-pneumonia is associated with lung immune cell infiltration and extensive proinflammatory cytokine secretion. Our previous study established that M. hyopneumoniae disrupts the host unfolded protein response (UPR), a process vital for the survival and immune function of macrophages. In this study, we demonstrated that M. hyopneumoniae targets the UPR- and caspase-12-mediated endoplasmic reticulum (ER)-associated classical intrinsic apoptotic pathway to interfere with host cell apoptosis signaling, thereby preserving the survival of host tracheal epithelial cells (PTECs) and alveolar macrophages (PAMs) during the early stages of infection. Even in the presence of apoptosis inducers, host cells infected with M. hyopneumoniae exhibited an anti-apoptotic potential. Further analyses revealed that M. hyopneumoniae suppresses the three UPR branches and their induced apoptosis. Interestingly, while UPR activation typically drives host macrophages toward an M2 polarization phenotype, M. hyopneumoniae specifically obstructs this process to maintain a proinflammatory phenotype in the host macrophages. Overall, our findings propose that M. hyopneumoniae inhibits the host UPR to sustain macrophage survival and a proinflammatory phenotype, which may be implicated in its pathogenesis in inducing host pneumonia.
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The immune response to Mycoplasma pneumoniae infection plays a key role in clinical symptoms. Previous investigations focused on the pro-inflammatory effects of leukocytes and the pivotal role of epithelial cell metabolic status in finely modulating the inflammatory response have been neglected. Herein, we examined how glycolysis in airway epithelial cells is affected by M. pneumoniae infection in an in vitro model. Additionally, we investigated the contribution of ATP to pulmonary inflammation. Metabolic analysis revealed a marked metabolic shift in bronchial epithelial cells during M. pneumoniae infection, characterized by increased glucose uptake, enhanced aerobic glycolysis, and augmented ATP synthesis. Notably, these metabolic alterations are orchestrated by adaptor proteins, MyD88 and TRAM. The resulting synthesized ATP is released into the extracellular milieu via vesicular exocytosis and pannexin protein channels, leading to a substantial increase in extracellular ATP levels. The conditioned medium supernatant from M. pneumoniae-infected epithelial cells enhances the secretion of both interleukin (IL)-1ß and IL-18 by peripheral blood mononuclear cells, partially mediated by the P2X7 purine receptor (P2X7R). In vivo experiments confirm that addition of a conditioned medium exacerbates pulmonary inflammation, which can be attenuated by pre-treatment with a P2X7R inhibitor. Collectively, these findings highlight the significance of airway epithelial aerobic glycolysis in enhancing the pulmonary inflammatory response and aiding pathogen clearance.
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Neumonía por Mycoplasma , Humanos , Mycoplasma pneumoniae , Leucocitos Mononucleares/metabolismo , Medios de Cultivo Condicionados , Células Epiteliales/microbiología , Pulmón/metabolismo , Interleucina-1beta/metabolismo , Adenosina TrifosfatoRESUMEN
BACKGROUND: Differences in opinion concerning the contribution of M. genitalium to pelvic inflammatory disease (PID) has resulted in inconsistencies across global testing and treatment guidelines. We conducted a systematic review and meta-analysis to determine the association between M. genitalium and PID and M. genitalium positivity within PID cases to provide a contemporary evidence base to inform clinical practice (PROSPERO registration: CRD42022382156). METHODS: PubMed, Embase, Medline and Web of Science were searched to Dec 1, 2023 for studies that assessed women for PID using established clinical criteria and used nucleic acid amplification tests to detect M. genitalium. We calculated summary estimates of the 1) association of M. genitalium with PID (pooled odds ratio [OR]) and 2) proportion of PID cases with M. genitalium detected (pooled M. genitalium positivity in PID), using random-effects meta-analyses, with 95% confidence intervals (CI). RESULTS: Nineteen studies were included: 10 estimated M. genitalium association with PID, and 19 estimated M. genitalium positivity in PID. M. genitalium infection was significantly associated with PID (pooled OR=1.67 [95%CI: 1.24-2.24]). The pooled positivity of M. genitalium in PID was 10.3% [95%CI: 5.63-15.99]. Subgroup and meta-regression analyses showed that M. genitalium positivity in PID was highest in the Americas, in studies conducted in both inpatient and outpatient clinic settings, and in populations at high risk of sexually transmitted infections. CONCLUSIONS: M. genitalium was associated with a 67% increase in odds of PID and was detected in about one in ten clinical diagnoses of PID. These data support testing women for M. genitalium at initial PID diagnosis.
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We report a large-scale outbreak of Mycoplasma pneumoniae respiratory infections encompassing 218 cases (0.8% of 26,449 patients tested) during 2023-2024 in Marseille, France. The bacterium is currently circulating and primarily affects children <15 years of age. High prevalence of co-infections warrants the use of a syndromic diagnostic strategy.
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Brotes de Enfermedades , Mycoplasma pneumoniae , Neumonía por Mycoplasma , Humanos , Francia/epidemiología , Neumonía por Mycoplasma/epidemiología , Neumonía por Mycoplasma/microbiología , Neumonía por Mycoplasma/historia , Adolescente , Niño , Preescolar , Masculino , Femenino , Adulto , Lactante , Adulto Joven , Persona de Mediana Edad , Historia del Siglo XXI , Anciano , Prevalencia , Coinfección/epidemiología , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/microbiologíaRESUMEN
During a 2023 outbreak of Mycoplasma pneumoniae-associated community-acquired pneumonia among children in northern Vietnam, we analyzed M. pneumoniae isolated from nasopharyngeal samples. In almost half (6 of 13) of samples tested, we found known A2063G mutations (macrolide resistance) and a novel C2353T variant on the 23S rRNA gene.
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Mutación , Mycoplasma pneumoniae , Neumonía por Mycoplasma , ARN Ribosómico 23S , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/clasificación , Humanos , Vietnam/epidemiología , ARN Ribosómico 23S/genética , Neumonía por Mycoplasma/microbiología , Neumonía por Mycoplasma/epidemiología , Neumonía por Mycoplasma/historia , Niño , Preescolar , Infecciones Comunitarias Adquiridas/microbiología , Infecciones Comunitarias Adquiridas/epidemiología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana/genéticaRESUMEN
Before the COVID-19 pandemic, Mycoplasma pneumoniae infections emerged during spring to summer yearly in Taiwan, but infections were few during the pandemic. M. pneumoniae macrolide resistance soared to 85.7% in 2020 but declined to 0% during 2022-2023. Continued molecular surveillance is necessary to monitor trends in macrolide-resistant M. pneumoniae.
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Antibacterianos , COVID-19 , Farmacorresistencia Bacteriana , Macrólidos , Mycoplasma pneumoniae , Neumonía por Mycoplasma , SARS-CoV-2 , Humanos , Taiwán/epidemiología , Macrólidos/farmacología , Macrólidos/uso terapéutico , Mycoplasma pneumoniae/efectos de los fármacos , Mycoplasma pneumoniae/genética , Neumonía por Mycoplasma/epidemiología , Neumonía por Mycoplasma/tratamiento farmacológico , Neumonía por Mycoplasma/microbiología , COVID-19/epidemiología , Niño , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Preescolar , Pandemias , Masculino , Femenino , Lactante , Adolescente , Pruebas de Sensibilidad MicrobianaRESUMEN
Mollicute infections, caused by Mycoplasma and Ureaplasma species, are serious complications after lung transplantation; however, understanding of the epidemiology and outcomes of these infections remains limited. We conducted a single-center retrospective study of 1156 consecutive lung transplants performed from 2010-2019. We used log-binomial regression to identify risk factors for infection and analyzed clinical management and outcomes. In total, 27 (2.3%) recipients developed mollicute infection. Donor characteristics independently associated with recipient infection were age ≤40 years (prevalence rate ratio [PRR] 2.6, 95% CI 1.0-6.9), White race (PRR 3.1, 95% CI 1.1-8.8), and purulent secretions on donor bronchoscopy (PRR 2.3, 95% CI 1.1-5.0). Median time to diagnosis was 16 days posttransplant (IQR: 11-26 days). Mollicute-infected recipients were significantly more likely to require prolonged ventilatory support (66.7% vs 21.4%), undergo dialysis (44.4% vs 6.3%), and remain hospitalized ≥30 days (70.4% vs 27.4%) after transplant. One-year posttransplant mortality in mollicute-infected recipients was 12/27 (44%), compared to 148/1129 (13%) in those without infection (P <.0001). Hyperammonemia syndrome occurred in 5/27 (19%) mollicute-infected recipients, of whom 3 (60%) died within 10 weeks posttransplant. This study highlights the morbidity and mortality associated with mollicute infection after lung transplantation and the need for better screening and management protocols.
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Trasplante de Pulmón , Mycoplasma , Infecciones por Ureaplasma , Humanos , Adulto , Estudios Retrospectivos , Infecciones por Ureaplasma/epidemiología , Infecciones por Ureaplasma/etiología , Infecciones por Ureaplasma/diagnóstico , Trasplante de Pulmón/efectos adversos , Trasplante de Pulmón/métodos , Factores de RiesgoRESUMEN
Mycoplasma hominis and Ureaplasma species are urogenital mollicutes that can cause serious donor-derived infections in lung transplant recipients. Best practices for mollicute screening remain unknown. We conducted a single-center prospective study analyzing lung transplants performed from October 5, 2020, to September 25, 2021, whereby donor and recipient bronchoalveolar lavage (BAL) samples obtained at time of transplant underwent mollicute screening via culture and polymerase chain reaction (PCR). Of 115 total lung transplants performed, 99 (86%) donors underwent combined mollicute BAL culture and PCR testing. The study cohort included these 99 donors and their matched recipients. In total, 18 (18%) of 99 donors screened positive via culture or PCR. Among recipients, 92 (93%) of 99 had perioperative BAL screening performed, and only 3 (3%) had positive results. After transplant, 9 (9%) recipients developed mollicute infection. Sensitivity of donor screening in predicting recipient mollicute infection was 67% (6/9) via culture and 56% (5/9) via PCR. Positive predictive value for donor culture was 75% (6/8), compared with 33% (5/15) for PCR. Donor screening via culture predicted all serious recipient mollicute infections and had better positive predictive value than PCR; however, neither screening test predicted all mollicute infections. Independent of screening results, clinicians should remain suspicious for posttransplant mollicute infection.
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OBJECTIVE(S): The emergence of antibiotic resistance has led to suboptimal treatment outcomes for Mycoplasma pneumoniae pneumonia (MPP). Exploring naturally occurring drug components that are both effective against MPP and non-toxic may be a promising choice. This study aimed to investigate the therapeutic effect of andrographolide nanoparticles on pneumonia caused by Mycoplasma pneumoniae infection. METHODS: Andrographolide alginate-poloxamer nanoparticles (AND-ALG-POL/NPs) were obtained by wet medium grinding, and the characterization and in vitro release of the prepared andrographolide nanoparticles were examined by high performance liquid chromatography, particle size analyzer, zeta potential meter and transmission electron microscopy. The cytotoxicity and anti-inflammatory effects of AND-ALG-POL/NPs were evaluated in vitro by MP-infected lung epithelial cells BEAS-2B. Symptoms of pneumonia, total cell count, total protein content and inflammatory factor levels in BALF were assessed by MP-induced pneumonia in BALB/c mice treated with AND-ALG-POL/NPs, and histopathological studies were performed on lung tissues from experimental animals. RESULTS: The results showed that the prepared AND-ALG-POL/NPs were homogeneous spherical with a diameter of 180 ± 23 nm, a zeta potential of (-14.4 ± 2.1) mV, an average encapsulation rate of 87.74 ± 0.87 %, and an average drug loading of 13.17 ± 0.54 %. AND-ALG-POL/NPs were capable of slow release in vitro and showed significant inhibitory ability against MP (P < 0.001). However, AND-ALG-POL/NPs were not cytotoxic to normal cells and alleviated MP infection-induced apoptosis and elevated inflammatory factors. In the in vivo experiments, AND-ALG-POL/NPs alleviated the symptoms of pneumonia in MPP mice, reduced the abnormally elevated total cell count, total protein content and inflammatory factor levels in BALF, and alleviated lung tissue edema, inflammatory cell infiltration and apoptosis (P < 0.001). Meanwhile, the therapeutic effects of AND-ALG-POL/NPs on MPP were similar to those of azithromycin (AZM) and higher than those of andrographolide (AND) free monotherapy (P < 0.001). CONCLUSION: In summary, the prepared AND-ALG-POL/NPs can effectively inhibit MPP in vitro and in vivo, and the effect is similar to that of AZM. Therefore, AND- ALG - POL/NPs have the potential to replace AZM as a potential drug for the treatment of MPP.
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Diterpenos , Nanopartículas , Neumonía por Mycoplasma , Ratones , Animales , Neumonía por Mycoplasma/tratamiento farmacológico , Mycoplasma pneumoniae , Pulmón/metabolismo , Nanopartículas/química , AzitromicinaRESUMEN
Mycoplasma pneumoniae (MP),as the most commonly infected respiratory pathogen in community-acquired pneumonia in preschool children,has becoming a prominent factor affecting children's respiratory health.Currently, there is a lack of easy, rapid, and accurate laboratory testing program for MP infection, which causes comparatively difficulty for clinical diagnostic.Here,we utilize loop-mediated isothermal amplification (LAMP) to amplify and characterize the P1 gene of MP, combined with nucleic acid lateral flow (NALF) for fast and visuallized detection of MP.Furthermore, we evaluated and analyzed the sensitivity, specificity and methodological consistency of the method.The results showed that the limit of detection(LoD) of MP-LAMP-NALF assay was down to 100 copys per reaction and there was no cross-reactivity with other pathogens infected the respiratory system. The concordance rate between MP-LAMP-NALF assay with quantitative real-time PCR was 94.3 %,which exhibiting excellent testing performance.We make superior the turnaround time of the MP-LAMP-NALF assay, which takes only about 50 min. In addition, there is no need for precision instruments and no restriction on the laboratory site.Collectively, LAMP-NALF assay targeting the P1 gene for Mycoplasma pneumoniae detection was a easy, precise and visual test which could be widely applied in outpatient and emergency departments or primary hospitals.When further optimized, it could be used as "point-of-care testing" of pathogens or multiple testing for pathogens.
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Técnicas de Diagnóstico Molecular , Mycoplasma pneumoniae , Técnicas de Amplificación de Ácido Nucleico , Neumonía por Mycoplasma , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , Neumonía por Mycoplasma/diagnóstico , Neumonía por Mycoplasma/microbiología , Técnicas de Diagnóstico Molecular/métodos , Sensibilidad y Especificidad , Límite de Detección , ADN Bacteriano/genéticaRESUMEN
Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre-lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.
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Mycoplasma , Vacunas , Animales , Cabras , Mycoplasma/genética , Serina ProteasasRESUMEN
Antimicrobial susceptibility testing (AST) of human mycoplasmas using microdilution is time-consuming. In this study, we compared the performance of MICRONAUT-S plates (Biocentric-Bruker) designed for AST of Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma hominis with the results using the Clinical & Laboratory Standards Institute (CLSI) reference method. Then, we investigated the prevalence and mechanisms of resistance to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. The two methods were compared using 60 strains. For the resistance prevalence study, U. parvum-, U. urealyticum-, and M. hominis-positive clinical specimens were collected for 1 month each year in 22 French diagnostic laboratories. MICs were determined using the MICRONAUT-S plates. The tet(M) gene was screened using PCR, and fluoroquinolone resistance-associated mutations were screened using PCR and Sanger sequencing. Comparing the methods, 99.5% (679/680) MICs obtained using the MICRONAUT-S plates concurred with those obtained using the CLSI reference method. For 90 M. hominis isolates, the tetracycline, levofloxacin, and moxifloxacin resistance rates were 11.1%, 2.2%, and 2.2%, respectively, with no clindamycin resistance. For 248 U. parvum isolates, the levofloxacin and moxifloxacin resistance rates were 5.2% and 0.8%, respectively; they were 2.9% and 1.5% in 68 U. urealyticum isolates. Tetracycline resistance in U. urealyticum (11.8%) was significantly (P < 0.001) higher than in U. parvum (1.2%). No macrolide resistance was observed. Overall, the customized MICRONAUT-S plates are a reliable, convenient tool for AST of human mycoplasmas. Tetracycline and fluoroquinolone resistance remain limited in France. However, the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires monitoring. IMPORTANCE: Antimicrobial susceptibility testing of human urogenital mycoplasmas using the CLSI reference broth microdilution method is time-consuming and requires the laborious preparation of antimicrobial stock solutions. Here, we validated the use of reliable, convenient plates designed for antimicrobial susceptibility testing that allows the simultaneous determination of the MICs of eight antibiotics of interest. We then investigated the prevalence and mechanisms of resistance of each of these bacteria to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. We showed that the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires ongoing monitoring.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad Microbiana , Infecciones por Mycoplasma , Mycoplasma hominis , Infecciones por Ureaplasma , Ureaplasma urealyticum , Ureaplasma , Humanos , Mycoplasma hominis/efectos de los fármacos , Francia/epidemiología , Ureaplasma/efectos de los fármacos , Ureaplasma/genética , Antibacterianos/farmacología , Infecciones por Ureaplasma/microbiología , Infecciones por Ureaplasma/epidemiología , Infecciones por Mycoplasma/microbiología , Infecciones por Mycoplasma/epidemiología , Ureaplasma urealyticum/efectos de los fármacos , Ureaplasma urealyticum/genética , Prevalencia , Fluoroquinolonas/farmacología , Macrólidos/farmacologíaRESUMEN
Significant increases in rates of sexually transmitted infections (STIs) caused by Trichomonas vaginalis (TV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG) are occurring in the United States. We present results of a U.S. study examining the intersection of STIs and vaginitis. Among 1,051 women with diagnoses for the presence or absence of bacterial vaginosis (BV) and/or symptomatic vulvovaginal candidiasis (VVC), 195 (18.5%) had one or more STIs, including 101 (9.6%) with TV, 24 (2.3%) with CT, 9 (0.8%) with NG, and 93 (8.8%) with MG. STI prevalence in BV-positive women was 26.3% (136/518), significantly higher than STI prevalence of 12.5% (59/474) in BV-negative women (P < 0.0002). Unlike infections with CT or NG, solo infections of MG or TV were each significantly associated with a diagnosis of BV-positive/VVC-negative (OR 3.0751; 95% CI 1.5797-5.9858, P = 0.0113, and OR 2.873; 95% CI 1.5687-5.2619, P = 0.0017, respectively) and with mixed infections containing MG and TV (OR 3.4886; 95% CI 1.8901-6.439, P = 0.0042, and OR 3.1858; 95% CI 1.809-5.6103, P = 0.0014, respectively). TV and MG infection rates were higher in all Nugent score (NS) categories than CT and NG infection rates; however, both STIs had similar comparative prevalence ratios to CT in NS 6-10 vs NS 0-5 (CT: 3.06% vs 1.4%, 2.2-fold; MG: 10.7% vs 6.1%, 1.8-fold; TV: 14.5% vs 7.0%, 2.1-fold). NG prevalence was relatively invariant by the NS category. These results highlight the complexity of associations of STIs with two major causes of vaginitis and underscore the importance of STI testing in women seeking care for abnormal vaginal discharge and inflammation. IMPORTANCE: This study reports high rates for sexually transmitted infections (STIs) in women seeking care for symptoms of vaginitis and bacterial vaginosis, revealing highly complex associations of STIs with two of the major causes of vaginal dysbiosis. These results underscore the importance of STI testing in women seeking care for abnormal vaginal discharge and inflammation.
RESUMEN
Concurrent infections with multiple pathogens are often described in cattle with respiratory illness. However, how the host-pathogen interactions influence the clinical outcome has been only partially explored in this species. Influenza D virus (IDV) was discovered in 2011. Since then, IDV has been detected worldwide in different hosts. A significant association between IDV and bacterial pathogens in sick cattle was shown in epidemiological studies, especially with Mycoplasma bovis. In an experimental challenge, IDV aggravated M. bovis-induced pneumonia. However, the mechanisms through which IDV drives an increased susceptibility to bacterial superinfections remain unknown. Here, we used the organotypic lung model precision-cut lung slices to study the interplay between IDV and M. bovis coinfection. Our results show that a primary IDV infection promotes M. bovis superinfection by increasing the bacterial replication and the ultrastructural damages in lung pneumocytes. In our model, IDV impaired the innate immune response triggered by M. bovis by decreasing the expression of several proinflammatory cytokines and chemokines that are important for immune cell recruitment and the bacterial clearance. Stimulations with agonists of cytosolic helicases and Toll-like receptors (TLRs) revealed that a primary activation of RIG-I/MDA5 desensitizes the TLR2 activation, similar to what was observed with IDV infection. The cross talk between these two pattern recognition receptors leads to a nonadditive response, which alters the TLR2-mediated cascade that controls the bacterial infection. These results highlight innate immune mechanisms that were not described for cattle so far and improve our understanding of the bovine host-microbe interactions and IDV pathogenesis. IMPORTANCE Since the spread of the respiratory influenza D virus (IDV) infection to the cattle population, the question about the impact of this virus on bovine respiratory disease (BRD) remains still unanswered. Animals affected by BRD are often coinfected with multiple pathogens, especially viruses and bacteria. In particular, viruses are suspected to enhance secondary bacterial superinfections. Here, we use an ex vivo model of lung tissue to study the effects of IDV infection on bacterial superinfections. Our results show that IDV increases the susceptibility to the respiratory pathogen Mycoplasma bovis. In particular, IDV seems to activate immune pathways that inhibit the innate immune response against the bacteria. This may allow M. bovis to increase its proliferation and to delay its clearance from lung tissue. These results suggest that IDV could have a negative impact on the respiratory pathology of cattle.