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BACKGROUND: Polymyxin B and E (colistin) have been pivotal in the treatment of XDR Gram-negative bacterial infections; however, resistance has emerged. A structurally related lipopeptide, octapeptin C4, has shown significant potency against XDR bacteria, including polymyxin-resistant strains, but its mode of action remains undefined. OBJECTIVES: We sought to compare and contrast the acquisition of resistance in an XDR Klebsiella pneumoniae (ST258) clinical isolate in vitro with all three lipopeptides to potentially unveil variations in their mode of action. METHODS: The isolate was exposed to increasing concentrations of polymyxins and octapeptin C4 over 20 days. Day 20 strains underwent WGS, complementation assays, antimicrobial susceptibility testing and lipid A analysis. RESULTS: Twenty days of exposure to the polymyxins resulted in a 1000-fold increase in the MIC, whereas for octapeptin C4 a 4-fold increase was observed. There was no cross-resistance observed between the polymyxin- and octapeptin-resistant strains. Sequencing of polymyxin-resistant isolates revealed mutations in previously known resistance-associated genes, including crrB, mgrB, pmrB, phoPQ and yciM, along with novel mutations in qseC. Octapeptin C4-resistant isolates had mutations in mlaDF and pqiB, genes related to phospholipid transport. These genetic variations were reflected in distinct phenotypic changes to lipid A. Polymyxin-resistant isolates increased 4-amino-4-deoxyarabinose fortification of lipid A phosphate groups, whereas the lipid A of octapeptin C4-resistant strains harboured a higher abundance of hydroxymyristate and palmitoylate. CONCLUSIONS: Octapeptin C4 has a distinct mode of action compared with the polymyxins, highlighting its potential as a future therapeutic agent to combat the increasing threat of XDR bacteria.
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Antibacterianos/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana Múltiple , Klebsiella pneumoniae/efectos de los fármacos , Lipopéptidos/farmacología , Péptidos Cíclicos/farmacología , Polimixina B/farmacología , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Mutación , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Rapid diagnosis and appropriate treatment is imperative in bacterial sepsis due increasing risk of mortality with every hour without appropriate antibiotic therapy. Atypical infections with fastidious organisms may take more than 4 days to diagnose leading to calls for improved methods for rapidly diagnosing sepsis. Capnocytophaga canimorsus is a slow-growing, fastidious gram-negative bacillus which is a common commensal within the mouths of dogs, but rarely cause infections in humans. C. canimorsus sepsis risk factors include immunosuppression, alcoholism and elderly age. Here we report on the application of emerging nanopore sequencing methods to rapidly diagnose an atypical case of C. canimorsus septic shock. CASE PRESENTATION: A 62 year-old female patient was admitted to an intensive care unit with septic shock and multi-organ failure six days after a reported dog bite. Blood cultures were unable to detect a pathogen after 3 days despite observed intracellular bacilli on blood smears. Real-time nanopore sequencing was subsequently employed on whole blood to detect Capnocytophaga canimorsus in 19 h. The patient was not immunocompromised and did not have any other known risk factors. Whole-genome sequencing of clinical sample and of the offending dog's oral swabs showed near-identical C. canimorsus genomes. The patient responded to antibiotic treatment and was discharged from hospital 31 days after admission. CONCLUSIONS: Use of real-time nanopore sequencing reduced the time-to-diagnosis of Capnocytophaga canimorsus in this case from 6.25 days to 19 h. Capnocytophaga canimorsus should be considered in cases of suspected sepsis involving cat or dog contact, irrespective of the patient's known risk factors.
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Mordeduras y Picaduras/complicaciones , Capnocytophaga/aislamiento & purificación , Choque Séptico/diagnóstico , Animales , Antibacterianos/uso terapéutico , Capnocytophaga/efectos de los fármacos , Capnocytophaga/genética , Gatos , Perros , Femenino , Infecciones por Bacterias Gramnegativas/diagnóstico , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Huésped Inmunocomprometido , Persona de Mediana Edad , Nanoporos , Análisis de Secuencia de ADN , Choque Séptico/inmunología , Choque Séptico/microbiologíaRESUMEN
Motivation: PE/PPE proteins, highly abundant in the Mycobacterium genome, play a vital role in virulence and immune modulation. Understanding their functions is key to comprehending the internal mechanisms of Mycobacterium. However, a lack of dedicated resources has limited research into PE/PPE proteins. Results: Addressing this gap, we introduce MycobactERIal PE/PPE proTeinS (MERITS), a comprehensive 3D structure database specifically designed for PE/PPE proteins. MERITS hosts 22 353 non-redundant PE/PPE proteins, encompassing details like physicochemical properties, subcellular localization, post-translational modification sites, protein functions, and measures of antigenicity, toxicity, and allergenicity. MERITS also includes data on their secondary and tertiary structure, along with other relevant biological information. MERITS is designed to be user-friendly, offering interactive search and data browsing features to aid researchers in exploring the potential functions of PE/PPE proteins. MERITS is expected to become a crucial resource in the field, aiding in developing new diagnostics and vaccines by elucidating the sequence-structure-functional relationships of PE/PPE proteins. Availability and implementation: MERITS is freely accessible at http://merits.unimelb-biotools.cloud.edu.au/.
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Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.
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The genome of Mycobacterium tuberculosis contains a relatively high percentage (10%) of genes that are poorly characterised because of their highly repetitive nature and high GC content. Some of these genes encode proteins of the PE/PPE family, which are thought to be involved in host-pathogen interactions, virulence, and disease pathogenicity. Members of this family are genetically divergent and challenging to both identify and classify using conventional computational tools. Thus, advanced in silico methods are needed to identify proteins of this family for subsequent functional annotation efficiently. In this study, we developed the first deep learning-based approach, termed Digerati, for the rapid and accurate identification of PE and PPE family proteins. Digerati was built upon a multipath parallel hybrid deep learning framework, which equips multi-layer convolutional neural networks with bidirectional, long short-term memory, equipped with a self-attention module to effectively learn the higher-order feature representations of PE/PPE proteins. Empirical studies demonstrated that Digerati achieved a significantly better performance (â¼18-20%) than alignment-based approaches, including BLASTP, PHMMER, and HHsuite, in both prediction accuracy and speed. Digerati is anticipated to facilitate community-wide efforts to conduct high-throughput identification and analysis of PE/PPE family members. The webserver and source codes of Digerati are publicly available at http://web.unimelb-bioinfortools.cloud.edu.au/Digerati/.
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Aprendizaje Profundo , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/genética , Virulencia/genéticaRESUMEN
A new variant of Streptococcus pyogenes serotype M1 (designated 'M1UK') has been reported in the United Kingdom, linked with seasonal scarlet fever surges, marked increase in invasive infections, and exhibiting enhanced expression of the superantigen SpeA. The progenitor S. pyogenes 'M1global' and M1UK clones can be differentiated by 27 SNPs and 4 indels, yet the mechanism for speA upregulation is unknown. Here we investigate the previously unappreciated expansion of M1UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1UK sub-lineages circulating in Australia also contain a novel toxin repertoire associated with epidemic scarlet fever causing S. pyogenes in Asia. A single SNP in the 5' transcriptional leader sequence of the transfer-messenger RNA gene ssrA drives enhanced SpeA superantigen expression as a result of ssrA terminator read-through in the M1UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance.
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Escarlatina , Infecciones Estreptocócicas , Humanos , Streptococcus pyogenes/genética , Escarlatina/epidemiología , Superantígenos , Proteínas Bacterianas/genética , Reino Unido , Exotoxinas/genética , Mutación , AustraliaRESUMEN
Mycobacterium tuberculosis genome comprises approximately 10% of two families of poorly characterised genes due to their high GC content and highly repetitive nature. The largest sub-group, the proline-glutamic acid polymorphic guanine-cytosine-rich sequence (PE_PGRS) family, is thought to be involved in host response and disease pathogenicity. Due to their high genetic variability and complexity of analysis, they are typically disregarded for further research in genomic studies. There are currently limited online resources and homology computational tools that can identify and analyse PE_PGRS proteins. In addition, they are computational-intensive and time-consuming, and lack sensitivity. Therefore, computational methods that can rapidly and accurately identify PE_PGRS proteins are valuable to facilitate the functional elucidation of the PE_PGRS family proteins. In this study, we developed the first machine learning-based bioinformatics approach, termed PEPPER, to allow users to identify PE_PGRS proteins rapidly and accurately. PEPPER was built upon a comprehensive evaluation of 13 popular machine learning algorithms with various sequence and physicochemical features. Empirical studies demonstrated that PEPPER achieved significantly better performance than alignment-based approaches, BLASTP and PHMMER, in both prediction accuracy and speed. PEPPER is anticipated to facilitate community-wide efforts to conduct high-throughput identification and analysis of PE_PGRS proteins.
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Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.
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COVID-19 , Animales , COVID-19/genética , Células CACO-2 , Chlorocebus aethiops , Humanos , Poliadenilación , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , SARS-CoV-2 , Análisis de Secuencia de ARN , Células VeroRESUMEN
Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria (Staphylococcus aureus and Streptococcus pneumoniae) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC90 ≤ 0.06 µg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae, as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.
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Antiinfecciosos , Infecciones por Bacterias Grampositivas , Staphylococcus aureus Resistente a Meticilina , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antiinfecciosos/farmacología , Biopelículas , Glicopéptidos/farmacología , Glicopéptidos/uso terapéutico , Lipoglucopéptidos/uso terapéutico , Mamíferos , Ratones , Pruebas de Sensibilidad Microbiana , Streptococcus pneumoniae , Vancomicina/farmacología , Vancomicina/uso terapéuticoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses subgenomic RNA (sgRNA) to produce viral proteins for replication and immune evasion. We apply long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA upregulates earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of open reading frame 1ab (ORF1ab) containing nsp1 joins to ORF10, and the 3' untranslated region (UTR) upregulates at 48 h post-infection in human cell lines. We identify double-junction sgRNA containing both TRS-dependent and -independent junctions. We find multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA and that sgRNA modifications are stable across transcript clusters, host cells, and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle.
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COVID-19/genética , SARS-CoV-2/genética , Transcripción Genética/genética , Animales , Células CACO-2 , Línea Celular , Chlorocebus aethiops , Epigénesis Genética , Genoma Viral/genética , Humanos , Evasión Inmune , Sistemas de Lectura Abierta , ARN Viral/genética , Transcriptoma , Células Vero , Proteínas Virales/genéticaRESUMEN
Pandoraea fibrosis is a newly identified Gram-negative bacterial species that was isolated from the respiratory tract of an Australian cystic fibrosis patient. The complete assembled genome sequences of two consecutive isolates (second isolate collected 11 months after antibiotic treatment) from the same individual are presented here.
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BACKGROUND: Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utility of DNA and RNA for prediction of antibiotic susceptibility. However, the effectiveness of bacterial direct RNA sequencing and base-calling has not previously been investigated. This study interrogated the genome and transcriptome of 4 extensively drug-resistant (XDR) K. pneumoniae clinical isolates; however, further antimicrobial susceptibility testing identified 3 isolates as pandrug-resistant (PDR). RESULTS: The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kb). DNA sequencing detected most resistance genes (≥70%) within 2 hours of sequencing. Neural network-based base-calling of direct RNA achieved up to 86% identity rate, although ≤23% of reads could be aligned. Direct RNA sequencing (with â¼6 times slower pore translocation) was able to identify (within 10 hours) ≥35% of resistance genes, including those associated with resistance to aminoglycosides, ß-lactams, trimethoprim, and sulphonamide and also quinolones, rifampicin, fosfomycin, and phenicol in some isolates. Direct RNA sequencing also identified the presence of operons containing up to 3 resistance genes. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes. CONCLUSION: Overall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.
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Farmacorresistencia Bacteriana Múltiple , Klebsiella pneumoniae/genética , Secuenciación de Nanoporos/métodos , RNA-Seq/métodos , Genoma Bacteriano , Klebsiella pneumoniae/efectos de los fármacos , Secuenciación de Nanoporos/normas , RNA-Seq/normas , TranscriptomaRESUMEN
INTRODUCTION: The octapeptins are a family of cyclic lipopeptides first reported in the 1970s then largely ignored. At the time, their reported antibiotic activity against polymyxin-resistant bacteria was a curiosity. Today, the advent of widespread drug resistance in Gram-negative bacteria has prompted their 'rediscovery.' The paucity of new antibiotics in the clinical pipeline is coupled with a global spread of increasing antibiotic resistance, particularly to meropenem and polymyxins B and E (colistin). Areas covered: We review the original discovery of octapeptins, their recent first chemical syntheses, and their mode of action, then discuss their potential as a new class of antibiotics to treat extensively drug-resistant (XDR) Gram-negative infections, with direct comparisons to the closely related polymyxins. Expert commentary: Cyclic lipopeptides in clinical use (polymyxin antibiotics) have significant dose-limiting nephrotoxicity inherent to their chemotype. This toxicity has prevented improved polymyxin analogs from progressing to the clinic, and tainted the perception of lipopeptide antibiotics in general. We argue that the octapeptins are fundamentally different from the polymyxins, with a disparate mode of action, spectra of action against MDR and XDR bacteria and a superior preclinical safety profile. They represent early-stage candidates that can help prime the antibiotic discovery pipeline.
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Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Lipopéptidos/farmacología , Animales , Antibacterianos/efectos adversos , Antibacterianos/farmacología , Diseño de Fármacos , Descubrimiento de Drogas/métodos , Farmacorresistencia Bacteriana Múltiple , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Lipopéptidos/efectos adversos , Polimixinas/efectos adversos , Polimixinas/farmacologíaRESUMEN
Extensively drug-resistant Klebsiella pneumoniae (XDR-KP) infections cause high mortality and are disseminating globally. Identifying the genetic basis underpinning resistance allows for rapid diagnosis and treatment. XDR isolates sourced from Greece and Brazil, including 19 polymyxin-resistant and five polymyxin-susceptible strains, were subjected to whole genome sequencing. Seventeen of the 19 polymyxin-resistant isolates harboured variations upstream or within mgrB. The most common mutation identified was an insertion at nucleotide position 75 in mgrB via an ISKpn26-like element in the ST258 lineage and ISKpn13 in one ST11 isolate. Three strains acquired an IS1 element upstream of mgrB and another strain had an ISKpn25 insertion at 133 bp. Other isolates had truncations (C28STOP, Q30STOP) or a missense mutation (D29E) affecting mgrB. Complementation assays revealed all mgrB perturbations contributed to resistance. Missense mutations in phoQ (T281M, G385C) were also found to facilitate resistance. Several variants in phoPQ co-segregating with the ISKpn26-like insertion were identified as potential partial suppressor mutations. Three ST258 samples were found to contain subpopulations with different resistance-conferring mutations, including the ISKpn26-like insertion colonizing with a novel mutation in pmrB (P158R), both confirmed via complementation assays. These findings highlight the broad spectrum of chromosomal modifications which can facilitate and regulate resistance against polymyxins in K. pneumoniae.
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Cromosomas Bacterianos/genética , ADN Bacteriano/aislamiento & purificación , Farmacorresistencia Bacteriana/genética , Klebsiella pneumoniae/efectos de los fármacos , Polimixinas/farmacología , Antibacterianos/farmacología , Brasil , Colistina/farmacología , ADN Bacteriano/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Biblioteca de Genes , Genes Bacterianos , Variación Genética , Grecia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Mutación , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required. METHODS: The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry. RESULTS: Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly chondroidal with chondrocyte-like cells embedded within collagen type II- and X-rich matrix. This corresponded with upregulation of mRNA for cartilage markers Col2a1, sox9 and Comp. Osteophytes, though infrequent, were more prevalent in later disease. CONCLUSIONS: The inflammation-driven IVD destruction was shown to be a prerequisite for axial disease progression to osteoproliferation in the PGISp mouse. Osteoproliferation led to vertebral body deformity and fusion but was never seen concurrent with persistent inflammation, suggesting a sequential process. The findings support that early intervention with anti-inflammatory therapies will be needed to limit destructive processes and consequently prevent progression of AS.
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Modelos Animales de Enfermedad , Progresión de la Enfermedad , Osteogénesis/fisiología , Espondilitis Anquilosante/etiología , Espondilitis Anquilosante/patología , Animales , Femenino , Inflamación/complicaciones , Inflamación/patología , Ratones , Ratones Endogámicos BALB C , Ratones NoqueadosRESUMEN
Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment.