mSphere最新文献

{"title":"Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications.","authors":"Dominic Wooding, Kate Buist, Alessandra Romero-Ramirez, Helen Savage, Rachel Watkins, Daisy Bengey, Caitlin Greenland-Bews, Caitlin R Thompson, Nadia Kontogianni, Richard Body, Gail Hayward, Rachel L Byrne, Susan Gould, Christopher Myerscough, Barry Atkinson, Victoria Shaw, Bill Greenhalf, Emily Adams, Ana Cubas-Atienzar, Saye Khoo, Tom Fletcher, Thomas Edwards","doi":"10.1128/msphere.00304-24","DOIUrl":"10.1128/msphere.00304-24","url":null,"abstract":"<p><p>Clinical trials of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics often include virological secondary endpoints to compare viral clearance and viral load reduction between treatment and placebo arms. This is typically achieved using quantitative reverse-transcriptase PCR (RT-qPCR), which cannot differentiate replicant competent virus from non-viable virus or free RNA, limiting its utility as an endpoint. Culture-based methods for SARS-CoV-2 exist; however, these are often insensitive and poorly standardized for use as clinical trial endpoints. We report optimization of a culture-based approach evaluating three cell lines, three detection methods, and key culture parameters. We show that Vero-angiotensin-converting enzyme 2-transmembrane serine protease 2 cells in combination with RT-qPCR of culture supernatants from the first passage provides the greatest overall detection of Delta viral replication (22 of 32, 68.8%), being able to identify viable virus in 83.3% (20 of 24) of clinical samples with initial Ct values of <30. Likewise, we demonstrate that RT-qPCR using culture supernatants from the first passage of Vero human signaling lymphocytic activation molecule cells provides the highest overall detection of Omicron viral replication (9 of 31, 29%), detecting live virus in 39.1% (9 of 23) of clinical samples with initial Ct values of <25. This assessment demonstrates that combining RT-qPCR with virological endpoint analysis has utility in clinical trials of therapeutics for SARS-CoV-2; however, techniques may require optimization based on dominant circulating strain.</p><p><strong>Importance: </strong>RT-qPCR is commonly used for virological endpoints during clinical trials for antiviral therapy to determine the quantity and presence of virus in a sample. However, RT-qPCR identifies viral RNA and cannot determine if viable virus is present. Existing culture-based techniques for SARS-CoV-2 are insensitive and not sufficiently standardized to be employed as clinical study endpoints. The use of a culture system to monitor replicating viruses could mitigate the possibility of molecular techniques identifying viral RNA from inactive or lysed viral particles. The methodology optimized in this study for detecting infectious viruses may have application as a secondary virological endpoint in clinical trials of therapeutics for SARS-CoV-2 in addition to numerous research processes.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0030424"},"PeriodicalIF":3.7,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11580409/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Adaptation to zinc restriction in <i>Streptococcus agalactiae</i>: role of the ribosomal protein and zinc-importers regulated by AdcR.","authors":"M Melet, S Blanchet, P Barbarin, E A Maunders, S L Neville, V Rong, L Mereghetti, C A McDevitt, A Hiron","doi":"10.1128/msphere.00614-24","DOIUrl":"10.1128/msphere.00614-24","url":null,"abstract":"<p><p>Zinc (Zn) is an essential cofactor for numerous bacterial proteins and altering Zn availability is an important component of host innate immunity. During infection, adaptation to both Zn deprivation and excess is critical for pathogenic bacteria development. To understand the adaptive responses to Zn availability of <i>Streptococcus agalactiae</i>, a pathogen causing invasive infections of neonates, global transcriptional profiling was conducted. Results highlight that in response to Zn limitation, genes belonging to the AdcR regulon, the master regulator of Zn homeostasis in streptococci, were overexpressed. Through a combination of <i>in silico</i> analysis and experimental validation, new AdcR-regulated targets were identified. Among them, we identified a duplicated ribosomal protein, RpsNb, and an ABC transporter, and examined the role of these genes in bacterial growth under Zn-restricted conditions. Our results indicated that, during Zn restriction, both the RpsNb protein and a potential secondary Zn transporter are important for <i>S. agalactiae</i> adaptation to Zn deficiency.</p><p><strong>Importance: </strong><i>Streptococcus agalactiae</i> is a bacterial human pathobiont causing invasive diseases in neonates. Upon infection, <i>S. agalactiae</i> is presented with Zn limitation and excess but the genetic systems that allow bacterial adaptation to these conditions remain largely undefined. A comprehensive analysis of <i>S. agalactiae</i> global transcriptional response to Zn availability shows that this pathogen manages Zn limitation mainly through upregulation of the AdcR regulon. We demonstrate that several AdcR-regulated genes are important for bacterial growth during Zn deficiency, including human biological fluids. Taken together, these findings reveal new mechanisms of <i>S. agalactiae</i> adaptation under conditions of metal deprivation.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0061424"},"PeriodicalIF":3.7,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11580457/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546501","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analytical measuring interval, linearity, and precision of serology assays for detection of SARS-CoV-2 antibodies according to CLSI guidelines.","authors":"Katarzyna Haynesworth, Troy J Kemp, Sarah A Loftus, Jordan Metz, Nicholas C Castro, Jimmie Bullock, David Fetterer, Ligia A Pinto","doi":"10.1128/msphere.00393-24","DOIUrl":"10.1128/msphere.00393-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Serology testing is commonly used to evaluate the immunogenicity of COVID-19 vaccines and measure antibodies as a marker of previous infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, four laboratory-developed serology enzyme-linked immunosorbent assays (SARS-CoV-2 anti-Spike and anti-Nucleocapsid immunoglobin G [IgG] and immunoglobin M [IgM]) calibrated to the WHO International Standard 20/136 were validated via analytical measuring interval (limit of blank [LOB], limit of detection [LOD], and limit of quantification [LOQ]), linearity, and precision according to the Clinical and Laboratory Standards Institute (CLSI) guidelines EP17-A2, EP06 2nd Edition, and EP05-A3. For Spike IgG, LOB was 3.0 binding antibody units per milliliter (BAU/mL), LOD was 4.1 BAU/mL, and LOQ was 27.1 BAU/mL. For Nucleocapsid IgG, LOB was 1.9 BAU/mL, LOD was 3.2 BAU/mL, and LOQ was 24.6 BAU/mL. For Spike IgM, LOB was 57.1 BAU/mL, LOD was 69.0 BAU/mL, and LOQ was 113.5 BAU/mL. For Nucleocapsid IgM, LOD was 242.2 BAU/mL, LOD was 289.9 BAU/mL, and LOQ was 572.4 BAU/mL. Each assay displayed good linearity (max % deviation from linearity (≥LOQ) = 10.7%). The result of within-run repeatability evaluation for medium positive samples was 7.7% for Spike IgG, 4.6% for Nucleocapsid IgG, 7.5% for Spike IgM, and 10.1% for Nucleocapsid IgM. The total precision, including medium positive sample variability across 20 days, three reagent kits, and two operators, was 13.5% for Spike IgG, 14.5% for Nucleocapsid IgG, 17.6% for Spike IgM, and 16.2% for Nucleocapsid IgM. The assays were successfully validated following the applicable CLSI guidelines. All assays met the ±20% deviation from linearity and the ±20% coefficient of variation specification for precision and repeatability.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;Reliable and validated serology assays are of increasing importance as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus continues to evolve and cause outbreaks. Validation of serology assays along with calibration to the International and National Standards (such as anti-SARS-CoV-2 Immunoglobulin WHO International Standard 20/136 or Frederick National Laboratory for Cancer Research's National Serology Standard COVID-NS01097) is critical to ensuring that results from clinical studies are reliable and comparable among various assays and laboratories. We describe the design and execution of a comprehensive study that established the analytical measuring intervals, linearity, precision, and repeatability of four in-house developed serology enzyme-linked immunosorbent assays (SARS-CoV-2 anti-Spike immunoglobin G [IgG] and immunoglobin M [IgM] and anti-Nucleocapsid IgG and IgM) following applicable Clinical and Laboratory Standards Institute (CLSI) guidelines. Overall, this study provides practical guidance on experimental design strategies and data analysis techniques, pertaining to the validation of COVID-19 serology","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0039324"},"PeriodicalIF":3.7,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11580426/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effects of global change drivers on the expression of pathogenicity and stress genes in dryland soil fungi.","authors":"Adriana L Romero-Olivares, Andrea Lopez, Jovani Catalan-Dibene, Scott Ferrenberg, Samuel E Jordan, Brooke Osborne","doi":"10.1128/msphere.00658-24","DOIUrl":"10.1128/msphere.00658-24","url":null,"abstract":"<p><p>The impacts of global climate change on dryland fungi have been understudied even though fungi are extremely sensitive to changes in the environment. Considering that many fungi are pathogens of plants and animals, including humans, their responses to anthropogenic change could have important implications for public health and food security. In this study, we investigated the potential physiological responses (i.e., metatranscriptomics) of pathogenicity and stress in dryland fungi exposed to global change drivers, drought, and the physical disturbance associated with land use. Specifically, we wanted to assess if there was an increase in the transcription of genes associated to pathogenicity and stress in response to global change drivers. In addition, we wanted to investigate which pathogenicity and stress genes were consistently differentially expressed under the different global change conditions across the heterogeneous landscape (i.e., microsite) of the Chihuahuan desert. We observed increased transcription of pathogenicity and stress genes, with specific genes being most upregulated in response to global change drivers. Additionally, climatic conditions linked to different microsites, such as those found under patches of vegetation, may play a significant role. We provide evidence supporting the idea that environmental stress caused by global change could contribute to an increase of pathogenicity as global climate changes. Specifically, increases in the transcription of stress and virulence genes, coupled with variations in gene expression, could lead to the onset of pathogenicity. Our work underscores the importance of studying dryland fungi exposed to global climate change and increases in existing fungal pathogens, as well as the emergence of new fungal pathogens, and consequences to public health and food security.</p><p><strong>Importance: </strong>The effects of global climate change on dryland fungi and consequences to our society have been understudied despite evidence showing that pathogenic fungi increase in abundance under global climate change. Moreover, there is a growing concern that global climate change will contribute to the emergence of new fungal pathogens. Yet, we do not understand what mechanisms might be driving this increase in virulence and the onset of pathogenicity. In this study, we investigate how fungi respond to global change drivers, physical disturbance, and drought, in a dryland ecosystem in terms of pathogenicity and stress. We find that indeed, under global change drivers, there is an increase in the transcription and expression of genes associated to pathogenicity and stress, but that microclimatic conditions matter. Our study shows the importance of investigating dryland fungi exposed to global climate change and impacts on our society, which may include threats to public health and food security.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0065824"},"PeriodicalIF":3.7,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11580470/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546507","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unraveling the role of cAMP signaling in <i>Giardia</i>: insights into PKA-mediated regulation of encystation and subcellular interactions.","authors":"Han-Wei Shih, Germain C M Alas, Alexander R Paredez","doi":"10.1128/msphere.00723-24","DOIUrl":"10.1128/msphere.00723-24","url":null,"abstract":"<p><p>cAMP plays an important role as a second messenger in the stage transition of various protozoan parasites. This signaling pathway relies on multiple effectors, such as protein kinase A (PKA), exchange protein activated by cAMP, and cAMP-response element binding protein transcription factors, to initiate signal transduction in humans. The <i>Giardia</i> genome only contains two adenylate cyclases (ACs), a single phosphodiesterase (PDE) and a single known PKA effector, and the specific functions of these components are not fully understood. In our previous research, we demonstrated the important role of AC2-dependent cAMP signaling in promoting the encystation program. Using the NanoBit technology, we emphasized the significance of AC2-dependent cAMP biosynthesis in regulating the dissociation of the PKA regulatory domain (PKAr) and PKA catalytic domain (PKAc). In this study, our objectives are twofold: first, we used the newly developed Split-Halo to examine subcellular interactions of <i>Gl</i>PKAr and <i>Gl</i>PKAc in <i>Giardia</i>; and second, we investigated whether PKAc regulates encystation-specific proteins. Our findings revealed distinct subcellular locations where <i>Gl</i>PKAr and <i>Gl</i>PKAc interacted during the trophozoite stage, including the flagella, basal bodies, and cytoplasm. Upon exposure to encystation stimuli, the interaction shifted from the flagella to the cytosol. Knockdown of <i>Gl</i>PKAc resulted in the downregulation of encystation-specific genes, leading to the production of fewer viable and water-resistant cysts indicating a role for PKA in the transcriptional regulation of encystation. These discoveries contribute to a deeper understanding of the cAMP signaling pathway and its important role in governing <i>Giardia</i>'s encystation process.</p><p><strong>Importance: </strong>The precise timing of interactions and subcellular compartmentation play crucial roles in signal transduction. The co-immunoprecipitation assay (CO-IP) has long been utilized to validate protein-protein interactions; however, CO-IPs lack spatial and temporal resolutions. Our recent study used the NanoBit assay, which showcased the reversible protein-protein interaction between PKAr and PKAc in response to cAMP analogs and encystation stimuli. Expanding on this groundwork, this study employs the Split-Halo assay to uncover the subcellular compartments where the PKAr and PKAc protein-protein interactions take place and respond to encystation stimuli. Taken together, these molecular tools provide spatiotemporal information on the protein-protein interaction, which will be useful in the field.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0072324"},"PeriodicalIF":3.7,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11580427/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Neutrophil prime unique transcriptional responses in intestinal organoids during infection with nontyphoidal <i>Salmonella enterica</i> serovars.","authors":"Anna-Lisa E Lawrence, Ryan P Berger, David R Hill, Sha Huang, Veda K Yadagiri, Brooke Bons, Courtney Fields, Jason S Knight, Christiane E Wobus, Jason R Spence, Vincent B Young, Basel H Abuaita, Mary X O'Riordan","doi":"10.1128/msphere.00693-24","DOIUrl":"10.1128/msphere.00693-24","url":null,"abstract":"<p><p>Nontyphoidal strains of <i>Salmonella enterica</i> are a major cause of foodborne illnesses, and infection with these bacteria results in inflammatory gastroenteritis. Polymorphonuclear leukocytes (PMNs), also known as neutrophils, are a dominant immune cell type found at the site of infection in <i>Salmonella-</i>infected individuals, but how they regulate infection outcome is not well understood. Here, we used a co-culture model of primary human PMNs and human intestinal organoids to probe the role of PMNs during infection with two of the most prevalent <i>Salmonella</i> serovars: <i>Salmonella enterica</i> serovar Enteritidis and Typhimurium. Using a transcriptomics approach, we identified a dominant role for PMNs in mounting differential immune responses including production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides. We also identified specific gene sets that were induced by PMNs in response to Enteritidis or Typhimurium infection. By comparing host responses to these serovars, we uncovered differential regulation of host metabolic pathways particularly induction of cholesterol biosynthetic pathways during Typhimurium infection and suppression of RNA metabolism during Enteritidis infection. Together, these findings provide insight into the role of human PMNs in modulating different host responses to pathogens that cause similar disease in humans.IMPORTANCENontyphoidal serovars of <i>Salmonella enterica</i> are known to induce robust recruitment of polymorphonuclear leukocytes (PMNs) in the gut during early stages of infection, but the specific role of PMNs in regulating infection outcome of different serovars is poorly understood. Due to differences in human infection progression compared to small animal models, characterizing the role of PMNs during infection has been challenging. Here, we used a co-culture model of human intestinal organoids with human primary PMNs to study the role of PMNs during infection of human intestinal epithelium. Using a transcriptomics approach, we define PMN-dependent reprogramming of the host response to <i>Salmonella</i>, establishing a clear role in amplifying pro-inflammatory gene expression. Additionally, the host response driven by PMNs differed between two similar nontyphoidal <i>Salmonella</i> serovars. These findings highlight the importance of building more physiological infection models to replicate human infection conditions to study host responses specific to individual pathogens.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0069324"},"PeriodicalIF":3.7,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142676207","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antibiotic tolerance due to restriction of cAMP-Crp regulation by mannitol, a non-glucose-family PTS carbon source.","authors":"Weiwei Zhu, Miaomiao Chen, Xue Zhang, Jie Su, Xinyang Zhang, Yuejuan Nong, Bowen Wang, Weihong Guo, Yunxin Xue, Dai Wang, Yiqun Liao, Jianjun Niu, Yuzhi Hong, Karl Drlica, Xilin Zhao","doi":"10.1128/msphere.00772-24","DOIUrl":"https://doi.org/10.1128/msphere.00772-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Enzyme-IIA (EIIA&lt;sup&gt;Glc&lt;/sup&gt;, Crr) of the phosphotransferase system (PTS) connects the uptake of glucose-family sugars to the cAMP-Crp regulatory cascade; phosphorylated EIIA&lt;sup&gt;Glc&lt;/sup&gt; enhances cAMP-Crp activity, which then contributes to the antibiotic-mediated accumulation of reactive oxygen species (ROS) and cell death. Defects in PTS cause antibiotic and disinfectant tolerance. We report that mannitol, a carbon source whose uptake does not use EIIA&lt;sup&gt;Glc&lt;/sup&gt;, reduces antibiotic-mediated killing of &lt;i&gt;Escherichia coli&lt;/i&gt; without affecting antibiotic minimal inhibitory concentration. Thus, mannitol promotes antibiotic tolerance. The tolerance pathway was defined by the loss of ciprofloxacin lethality from the deletion of &lt;i&gt;ptsI&lt;/i&gt; (first gene in PTS), &lt;i&gt;mtlA&lt;/i&gt; (mannitol-specific Enzyme-II), &lt;i&gt;cyaA&lt;/i&gt; (cAMP synthase), and &lt;i&gt;crp&lt;/i&gt; (cAMP receptor protein) but not &lt;i&gt;crr&lt;/i&gt; (EIIA&lt;sup&gt;Glc&lt;/sup&gt;). A &lt;i&gt;crp*&lt;/i&gt; mutant, which encodes a constitutively active Crp that bypasses the need for cAMP activation, also decreased mannitol-mediated antibiotic tolerance, as did exogenous cAMP. Thus, inhibition of antibiotic lethality by mannitol involves both PTS-mediated mannitol uptake and suppression of cAMP-Crp action, independent of EIIA&lt;sup&gt;Glc&lt;/sup&gt;. Mannitol suppressed the downstream antibiotic-mediated transcription of genes involved in NADH production and cellular respiration, expression of a superoxide reporter gene (&lt;i&gt;soxS&lt;/i&gt;), and accumulation of antibiotic-mediated ROS. Similar phenomena were observed with mannose and sorbitol, demonstrating that non-glucose PTS carbon sources can cause antibiotic tolerance by a novel path that reduces the ROS-promoting activity of cAMP-Crp. The work emphasizes that antibiotic tolerance, which contributes to disease relapse and the need for prolonged antibiotic treatment, can result from commonly consumed carbohydrates. This finding, plus mutations that interfere specifically with antibiotic lethality, makes tolerance a high probability event.IMPORTANCEBacterial tolerance constitutes a significant threat to anti-infective therapy and potentially to the use of disinfectants. Deficiency mutations that reduce glucose uptake, central carbon metabolism, and cellular respiration confer antibiotic/disinfectant tolerance by reducing the accumulation of reactive metabolites, such as reactive oxygen species. We identified novel environmental generators of tolerance by showing that non-glucose carbohydrates, such as mannitol, mannose, and sorbitol, generate tolerance to multiple antibiotic classes. Finding that these sugars inhibit a universal, stress-mediated death pathway emphasizes the potential danger of compounds that block the lethal response to severe stress. Immediate practical importance derives from mannitol being a popular food sweetener, a treatment for glaucoma, and a dehydrating agent for treating cerebral edema, including cases caused by bacterial infection: antibiotic tolerance coul","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0077224"},"PeriodicalIF":3.7,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142676203","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Whole genome sequencing revealed high proportions of ST152 MRSA among clinical <i>Staphylococcus aureus</i> isolates from ten hospitals in Ghana.","authors":"Beverly Egyir, Christian Owusu-Nyantakyi, Alfred Bortey, Grebstad Rabbi Amuasi, Felicia Amoa Owusu, William Boateng, Hawawu Ahmed, Justice Kwesi Danso, Agnes Akosua Gyamaah Oclu, Quaneeta Mohktar, Georgina Tetteh-Ocloo, Harold Amegbletor, Kwabena Fosu, Francis Kwame Morgan Tetteh, Solomon Asante-Sefa, Oliver Nangkuu Deberu, Kennedy Mensah Osei, Joana Twasam, Sarkodie Kodom, Esther Gyinae, James Sampah, Nicholas Dzifa Dayie, Noah Obeng-Nkrumah, William Addo Mills-Pappoe, Gifty Boateng, Pernille Nilsson, Harriet Affran Bonful, Bright Adu, Rene S Hendriksen","doi":"10.1128/msphere.00446-24","DOIUrl":"https://doi.org/10.1128/msphere.00446-24","url":null,"abstract":"<p><p>Previous studies in Ghana indicated low prevalence of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) and predominance of ST152 methicillin-susceptible <i>S. aureus</i> (MSSA) among clinical isolates. ST152 MRSA clones are associated with severe infections and epidemics. Using whole genome sequencing (WGS), 159 <i>S</i>. <i>aureus</i> isolated from clinical sources (wound, blood, urine, ear, abscess, umbilical cord, eye, vaginal samples, and others) from 10 hospitals across Ghana were investigated. <i>mecA</i> (gene for methicillin resistance) was detected in 38% of the isolates. Panton-Valentine leucocidin toxin (PVL) gene occurred in 65% isolates, with 84% of the MRSA's harboring the PVL gene. ST152 was the major clone, with 74% harboring the <i>mecA</i> gene. Other MRSA clones detected were ST5, ST5204, ST852, and ST1. MSSA clones included ST3249, ST152, ST5, ST1, and ST8. Twenty-three genes encoding resistance to 12 antimicrobial classes were observed with <i>blaZ</i> (97%) being the most prevalent. Other predominant resistance genes included <i>tetK</i> (46%), <i>cat</i> (42%), and <i>dfrG</i> (36%) encoding resistance for tetracyclines, phenicols, and diaminopyrimidine, respectively. Virulence genes for enterotoxins, biofilms, toxic-shock-syndrome toxins, hemolysins<i>,</i> and leukotoxins were also detected. Phylogenetic analysis revealed a shift in the dominant clone from MSSA ST152 to MRSA ST152 over the past decade. The study provides valuable insights into the genomic content of <i>S. aureus</i> from clinical sources in Ghana. The finding of ST152 MRSA in high numbers suggests a shifting epidemiological landscape of these pathogens and continuous surveillance using robust tools like WGS is needed to monitor the rise and spread of these epidemic clones in the country.IMPORTANCESince its emergence in 1959, MRSA has been a significant public health concern, causing infections in both clinical and community settings. Patients with MRSA-related infections experience higher mortality rates due to its ability to evade antimicrobials and immune defenses. In Ghana, understanding the molecular epidemiology of MRSA has been hindered by the lack of appropriate laboratory infrastructure and the limited capacity for molecular data analysis. This study, the largest genomic study of <i>S. aureus</i> in Ghana, addresses this gap by utilizing whole genome sequencing to examine the diversity of circulating <i>S. aureus</i> strains from 10 hospitals. Our findings highlight the predominance of pandemic clones, particularly ST152, and the notable transition of ST152 MSSA to ST152 MRSA over the past decade. The findings from this study supports AMR surveillance efforts in Ghana and emphasize the importance of implementing genomic surveillance using WGS to comprehensively monitor the rise and spread of multi-drug-resitant organisms such as MRSA in the country.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0044624"},"PeriodicalIF":3.7,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142676210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR-Cas9-based approaches for genetic analysis and epistatic interaction studies in <i>Coxiella burnetii</i>.","authors":"Samuel Steiner, Craig R Roy","doi":"10.1128/msphere.00523-24","DOIUrl":"10.1128/msphere.00523-24","url":null,"abstract":"<p><p><i>Coxiella burnetii</i> is an obligate intracellular bacterial pathogen that replicates to high numbers in an acidified lysosome-derived vacuole. Intracellular replication requires the Dot/Icm type IVB secretion system, which translocates over 100 different effector proteins into the host cell. Screens employing random transposon mutagenesis have identified several <i>C. burnetii</i> effectors that play an important role in intracellular replication; however, the difficulty in conducting directed mutagenesis has been a barrier to the systematic analysis of effector mutants and to the construction of double mutants to assess epistatic interactions between effectors. Here, two CRISPR-Cas9 technology-based approaches were developed to study <i>C. burnetii</i> phenotypes resulting from targeted gene disruptions. CRISPRi was used to silence gene expression and demonstrated that silencing of effectors or Dot/Icm system components resulted in phenotypes similar to those of transposon insertion mutants. A CRISPR-Cas9-mediated cytosine base editing protocol was developed to generate targeted loss-of-function mutants through the introduction of premature stop codons into <i>C. burnetii</i> genes. Cytosine base editing successfully generated double mutants in a single step. A double mutant deficient in both <i>cig57</i> and <i>cig2</i> had a robust and additive intracellular replication defect when compared to either single mutant, which is consistent with Cig57 and Cig2 functioning in independent pathways that both contribute to a vacuole that supports <i>C. burnetii</i> replication. Thus, CRISPR-Cas9-based technologies expand the genetic toolbox for <i>C. burnetii</i> and will facilitate genetic studies aimed at investigating the mechanisms this pathogen uses to replicate inside host cells.</p><p><strong>Importance: </strong>Understanding the genetic mechanisms that enable <i>C. burnetii</i> to replicate in mammalian host cells has been hampered by the difficulty in making directed mutations. Here, a reliable and efficient system for generating targeted loss-of-function mutations in <i>C. burnetii</i> using a CRISPR-Cas9-assisted base editing approach is described. This technology was applied to make double mutants in <i>C. burnetii</i> that enabled the genetic analysis of two genes that play independent roles in promoting the formation of vacuoles that support intracellular replication. This advance will accelerate the discovery of mechanisms important for <i>C. burnetii</i> host infection and disease.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0052324"},"PeriodicalIF":3.7,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142668534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Erratum for Longley et al., \"Signatures of Mollicutes-related endobacteria in publicly available Mucoromycota genomes\".","authors":"Reid Longley, Aaron J Robinson, Olivia A Asher, Earl Middlebrook, Gregory Bonito, Patrick S G Chain","doi":"10.1128/msphere.00881-24","DOIUrl":"https://doi.org/10.1128/msphere.00881-24","url":null,"abstract":"","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0088124"},"PeriodicalIF":3.7,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142648473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}