Microbiology spectrum最新文献

{"title":"Stability assessment of housekeeping genes for qRT-PCR in <i>Yersinia enterocolitica</i> cultured at 22°C and 37°C.","authors":"Chuchu Li, Lu Zhou, Xiaoxuan Ma, Liguo Zhu, Jia Li, Lingning Meng, Mei Han, Danwei Wang, Han Shen, Chang Liu","doi":"10.1128/spectrum.01146-24","DOIUrl":"10.1128/spectrum.01146-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;&lt;i&gt;Yersinia enterocolitica&lt;/i&gt;, a species within the genus &lt;i&gt;Yersinia&lt;/i&gt;, thrives optimally at 22-25°C but can also grow at the mammalian core body temperature of 37°C. This dual temperature adaptability necessitates establishing both temperature conditions in research to examine the effects on various biological processes. In quantitative real-time PCR (qRT-PCR) assays, the selection of appropriate housekeeping genes is vital for data accuracy. Nevertheless, the lack of alternatives and information often leads to the default use of the 16S rRNA gene despite potential limitations. This investigation sourced 16 potential reference genes through a comprehensive review of the literature and transcriptome sequencing data analysis. We validated the expression stability of these genes via qRT-PCR across 12 &lt;i&gt;Y. enterocolitica&lt;/i&gt; strains, representing the four prevalent serotypes O:3, O:5,27, O:8, and O:9, isolated from diarrheal patient stool samples. This approach aimed to minimize the impact of serotype heterogeneity. After acquiring Cq values, gene stability was evaluated using four established algorithms-ΔCq, geNorm, NormFinder, and BestKeeper-and subsequently synthesized into a consolidated ranking through the Robust Rank Aggregation (RRA) method. Our study suggests that the genes &lt;i&gt;glnS&lt;/i&gt;, &lt;i&gt;nuoB&lt;/i&gt;, &lt;i&gt;glmS&lt;/i&gt;, &lt;i&gt;gyrB&lt;/i&gt;, &lt;i&gt;dnaK&lt;/i&gt;, and &lt;i&gt;thrS&lt;/i&gt; maintain consistent expression across varying culture temperatures, supporting their candidacy as robust housekeeping genes. We advise against the exclusive use of 16S rRNA for this purpose. Should tradition prevail in its utilization, it must be employed with discernment, preferably alongside one or two of the housekeeping genes identified in this study as internal controls.IMPORTANCEIn our study, we focused on identifying stable reference genes for quantitative real-time PCR (qRT-PCR) experiments on &lt;i&gt;Y. enterocolitica&lt;/i&gt; cultured at different temperatures (22°C and 37°C). After thoroughly evaluating 16 candidate genes, we identified six genes-&lt;i&gt;glnS&lt;/i&gt;, &lt;i&gt;nuoB&lt;/i&gt;, &lt;i&gt;glmS&lt;/i&gt;, &lt;i&gt;gyrB&lt;/i&gt;, &lt;i&gt;dnaK&lt;/i&gt;, and &lt;i&gt;thrS&lt;/i&gt;-as exhibiting stable expression across these temperature conditions, making them ideal reference genes for &lt;i&gt;Y. enterocolitica&lt;/i&gt; studies. This discovery is crucial for ensuring the accuracy and reliability of qRT-PCR data, as the choice of appropriate reference genes is key to normalizing expression data and minimizing experimental variability. Importantly, our research extended beyond bioinformatics analysis by incorporating validation with clinical strains, bridging the gap between theoretical predictions and practical application. This approach not only underscores the robustness and reliability of our findings but also directly addresses the critical need for experimental validation in the field. By providing a set of validated, stably expressed reference genes, our work offers valuable guidance for designing experiments involving &lt;i&gt;Y. enterocolitica&lt;","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11536982/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142372338","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":"Identification and characterization of a novel small viral peptide (VSP59) encoded by <i>Bombyx mori</i> cypovirus (BmCPV) that negatively regulates viral replication.","authors":"Manman Cao, Qunnan Qiu, Xing Zhang, Wenxue Zhang, Zeen Shen, Chang Ma, Min Zhu, Jun Pan, Xingyu Tong, Guangli Cao, Chengliang Gong, Xiaolong Hu","doi":"10.1128/spectrum.00826-24","DOIUrl":"10.1128/spectrum.00826-24","url":null,"abstract":"<p><p><i>Bombyx mori</i> cypovirus (BmCPV), a member of the Reoviridae family, is a well-established research model for double-stranded RNA (dsRNA) viruses with segmented genomes. Despite its small genome size, the coding potential of BmCPV remains largely unexplored. In this study, we identified a novel small open reading frame within the S10 dsRNA genome, encoding a small viral peptide (VSP59) with 59 amino acid residues. Functional characterization revealed that VSP59 acts as a negative regulator of viral replication. VSP59 predominantly localizes to the cytoplasm, where it interacts with prohibitin 2 (PHB2), an inner membrane mitophagy receptor. This interaction targets mitochondria and triggers caspase 3-dependent apoptosis. Transient expression of <i>vsp59</i> in BmN cells suppressed viral replication, an effect that was reversed by silencing PHB2 expression. Moreover, recombinant BmCPV with a mutated <i>vsp59</i> exhibited reduced replication. Our findings demonstrate that VSP59 interacts with PHB2 on mitochondria, inducing apoptosis and thereby diminishing viral replication. This study expands our understanding of the genetic information encoded by the BmCPV genome and highlights the role of novel small peptides in host-virus interactions.</p><p><strong>Importance: </strong>A novel small open reading frame (sORF) from the viral genome was identified and characterized. The sORF could encode a small viral peptide (VSP59) that targeted mitochondria and induced prohibitin 2-related apoptosis, further attenuating <i>Bombyx mori</i> cypovirus replication.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537000/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391745","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":"Direct genome sequencing of respiratory viruses from low viral load clinical specimens using the target capture sequencing technology.","authors":"Nobuhiro Takemae, Yumani Kuba, Kunihiro Oba, Tsutomu Kageyama","doi":"10.1128/spectrum.00986-24","DOIUrl":"10.1128/spectrum.00986-24","url":null,"abstract":"<p><p>The use of metagenomic next-generation sequencing technology to obtain complete viral genome sequences directly from clinical samples with low viral load remains challenging-especially in the case of respiratory viruses-due to the low copy number of viral versus host genomes. To overcome this limitation, target capture sequencing for the enrichment of specific genomes has been developed and applied for direct genome sequencing of viruses. However, as the efficiency of enrichment varies depending on the probes, the type of clinical sample, etc., validation is essential before target capture sequencing can be applied to clinical diagnostics. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A. We focused on clinical specimens containing low copy numbers of viral genomes. Target capture sequencing yielded approximately 180- and 2,000-fold higher read counts of SARS-CoV-2 and influenza A virus, respectively, than metagenomic sequencing when the RNA extracted from specimens contained 59.3 copies/µL of SARS-CoV-2 or 625.1 copies/µL of influenza A virus. In addition, the target capture sequencing identified sequence reads in all SARS-CoV-2- or influenza type A-positive specimens with <26 RNA copies/µL, some of which also yielded >70% of the full-length genomes of SARS-CoV-2 or influenza A virus. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with viruses other than SARS-CoV-2, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.IMPORTANCETarget capture sequencing has been developed and applied for direct genome sequencing of viruses in clinical specimens to overcome the low detection sensitivity of metagenomic next-generation sequencing. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A, focusing on clinical specimens containing low copy numbers of viral genomes. Our results showed that the target capture sequencing yielded dramatically higher read counts than metagenomic sequencing for both viruses. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with other viruses, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537015/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470047","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":"Pre-challenge gut microbial signature predicts RhCMV/SIV vaccine efficacy in rhesus macaques.","authors":"Hayden N Brochu, Elise Smith, Sangmi Jeong, Michelle Carlson, Scott G Hansen, Jennifer Tisoncik-Go, Lynn Law, Louis J Picker, Michael Gale, Xinxia Peng","doi":"10.1128/spectrum.01285-24","DOIUrl":"10.1128/spectrum.01285-24","url":null,"abstract":"<p><p>Rhesus cytomegalovirus expressing simian immunodeficiency virus (RhCMV/SIV) vaccines protect ~59% of vaccinated rhesus macaques against repeated limiting-dose intra-rectal exposure with highly pathogenic SIVmac239M, but the exact mechanism responsible for the vaccine efficacy is unknown. It is becoming evident that complex interactions exist between gut microbiota and the host immune system. Here, we aimed to investigate if the rhesus gut microbiome impacts RhCMV/SIV vaccine-induced protection. Three groups of 15 rhesus macaques naturally pre-exposed to RhCMV were vaccinated with RhCMV/SIV vaccines. Rectal swabs were collected longitudinally both before SIV challenge (after vaccination) and post-challenge and were profiled using 16S rRNA based microbiome analysis. We identified ~2,400 16S rRNA amplicon sequence variants (ASVs), representing potential bacterial species/strains. Global gut microbial profiles were strongly associated with each of the three vaccination groups, and all animals tended to maintain consistent profiles throughout the pre-challenge phase. Despite vaccination group differences, by using newly developed compositional data analysis techniques, we identified a common gut microbial signature predictive of vaccine protection outcome across the three vaccination groups. Part of this microbial signature persisted even after SIV challenge. We also observed a strong correlation between this microbial signature and an early signature derived from whole blood transcriptomes in the same animals. Our findings indicate that changes in gut microbiomes are associated with RhCMV/SIV vaccine-induced protection and early host response to vaccination in rhesus macaques.IMPORTANCEThe human immunodeficiency virus (HIV) has infected millions of people worldwide. Unfortunately, still there is no vaccine that can prevent or treat HIV infection. A promising pre-clinical HIV vaccine based on rhesus cytomegalovirus (RhCMV) expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) provides sustained, durable protection against SIV challenge in ~59% of vaccinated rhesus macaques. There is an urgent need to understand the cause of this protection vs non-protection outcome. In this study, we profiled the gut microbiomes of 45 RhCMV/SIV vaccinated rhesus macaques and identified gut microbial signatures that were predictive of RhCMV/SIV vaccination groups and vaccine protection outcomes. These vaccine protection-associated microbial features were significantly correlated with early vaccine-induced host immune signatures in whole blood from the same animals. These findings show that the gut microbiome may be involved in RhCMV/SIV vaccine-induced protection, warranting further research into the impact of the gut microbiome in human vaccine trials.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537114/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142350147","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":"Insight into the outer membrane asymmetry of <i>P. aeruginosa</i> and the role of MlaA in modulating the lipidic composition, mechanical, biophysical, and functional membrane properties of the cell envelope.","authors":"M Kaur, N Mozaheb, T O Paiva, M-F Herent, F Goormaghtigh, A Paquot, R Terrasi, E Mignolet, J-L Décout, J H Lorent, Y Larondelle, G G Muccioli, J Quetin-Leclercq, Y F Dufrêne, M-P Mingeot-Leclercq","doi":"10.1128/spectrum.01484-24","DOIUrl":"10.1128/spectrum.01484-24","url":null,"abstract":"<p><p>In Gram-negative bacteria, the outer membrane (OM) is asymmetric, with lipopolysaccharides (LPS) in the outer leaflet and glycerophospholipids (GPLs) in the inner leaflet. The asymmetry is maintained by the Mla system (MlaA-MlaBCDEF), which contributes to lipid homeostasis by removing mislocalized GPLs from the outer leaflet of the OM. Here, we ascribed how <i>Pseudomonas aeruginosa</i> ATCC 27853 coordinately regulates pathways to provide defense against the threats posed by the deletion of <i>mlaA</i>. Especially, we explored (i) the effects on membrane lipid composition including LPS, GPLs, and lysophospholipids, (ii) the biophysical properties of the OM such as stiffness and fluidity, and (iii) the impact of these changes on permeability, antibiotic susceptibility, and membrane vesicles (MVs) generation. Deletion of <i>mlaA</i> induced an increase in total GPLs and a decrease in LPS level while also triggering alterations in lipid A structures (arabinosylation and palmitoylation), likely to be induced by a two-component system (PhoPQ-PmrAB). Altered lipid composition may serve a physiological purpose in regulating the mechanobiological and functional properties of <i>P. aerugino</i>sa. We demonstrated an increase in cell stiffness without alteration of turgor pressure and inner membrane (IM) fluidity in ∆<i>mlaA</i>. In addition, membrane vesiculation increased without any change in OM/IM permeability. An amphiphilic aminoglycoside derivative (3',6-dinonyl neamine) that targets <i>P. aeruginosa</i> membranes induced an opposite effect on ∆<i>mlaA</i> strain with a trend toward a return to the situation observed for the WT strain. Efforts dedicated to understanding the crosstalk between the OM lipid composition, and the mechanical behavior of bacterial envelope, is one needed step for designing new targets or new drugs to fight <i>P. aeruginosa</i> infections.IMPORTANCE<i>Pseudomonas aeruginosa</i> is a Gram-negative bacterium responsible for severe hospital-acquired infections. The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore, compromising this structure could increase sensitivity to antibiotics. The OM is asymmetric with the highly packed lipopolysaccharide monolayer at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla pathway resulting in the retrograde transport of glycerophospholipids from the OM to the inner membrane. In this study, we show that deleting <i>mlaA</i>, the membrane component of Mla system located at the OM, affects the mechanical and functional properties of <i>P. aeruginosa</i> cell envelope. Our results provide insights into the role of MlaA, involved in the Mla transport pathway in <i>P. aeruginosa</i>.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537012/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142381171","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":"Transcriptomics identify the triggering of citrate export as the key event caused by manganese deficiency in <i>Aspergillus niger</i>.","authors":"Erzsébet Fekete, Vivien Bíró, Alexandra Márton, István Bakondi-Kovács, Erzsébet Sándor, Béla Kovács, Nicholas Geoffrion, Adrian Tsang, Christian P Kubicek, Levente Karaffa","doi":"10.1128/spectrum.01906-24","DOIUrl":"10.1128/spectrum.01906-24","url":null,"abstract":"<p><p>For over a century, the filamentous Ascomycete fungus <i>Aspergillus niger</i> has played a pivotal role in the industrial production of citric acid. A critical fermentation parameter that sustains high-yield citric acid accumulation is the suboptimal concentration of manganese(II) ions in the culture broth at the early stages of the process. However, the requirement for this deficiency has not been investigated on a functional genomics level. In this study, we compared the transcriptome of the citric acid hyper-producer <i>A. niger</i> NRRL2270 strain grown under citric acid-producing conditions in 6-L scale bioreactors at Mn²⁺ ion-deficient (5 ppb) and Mn²⁺ ion-sufficient (100 ppb) conditions at three early time points of cultivation. Of the 11,846 genes in the genome, 963 genes (8.1% of the total) were identified as significantly differentially expressed under these conditions. Disproportionately high number of differentially regulated genes encode predicted extracellular and membrane proteins. The most abundant gene group that was upregulated in Mn²⁺ ion deficiency condition encodes enzymes acting on polysaccharides. In contrast, six clusters of genes encoding secondary metabolites showed downregulation under manganese deficiency. Mn²⁺ deficiency also triggers upregulation of the <i>cexA</i> gene, which encodes the citrate exporter. We provide functional evidence that the upregulation of <i>cexA</i> is caused by the intracellular accumulation of citrate or acetyl-CoA and is a major factor in triggering citrate overflow.</p><p><strong>Importance: </strong>Citric acid is produced on industrial scale by batch fermentation of the filamentous fungus <i>Aspergillus niger</i>. High-yield citric acid production requires a low (<5 ppb) manganese(II) ion concentration in the culture broth. However, the requirement for this deficiency has not been investigated on a functional genomics level. Here, we compared the transcriptome of a citric acid hyper-producer <i>A. niger</i> strain grown under citric acid-producing conditions in 6-L scale bioreactors at Mn²⁺ ion-deficient (5 ppb) and Mn²⁺ ion-sufficient (100 ppb) conditions at three early time points of cultivation. We observed that Mn²⁺ deficiency triggers an upregulation of the citrate exporter gene cexA and provides functional evidence that this event is responsible for citrate overflow. In addition to the industrial relevance, this is the first study that examined the role of Mn²⁺ ion deficiency in a heterotrophic eukaryotic cell on a genome-wide scale.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537073/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391757","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":"Investigation of <i>Citrobacter freundii</i> clinical isolates in a Chinese hospital during 2020-2022 revealed genomic characterization of an extremely drug-resistant <i>C. freundii</i> ST257 clinical strain GMU8049 co-carrying <i>bla</i>_NDM-1 and a novel <i>bla</i>_CMY variant.","authors":"Mujie Zhang, Zhiqiu Yin, Baozhu Chen, Zhanpeng Yu, Jiaxin Liang, Xiaoyan Tian, Defu Li, Xiaoyan Deng, Liang Peng","doi":"10.1128/spectrum.04254-23","DOIUrl":"10.1128/spectrum.04254-23","url":null,"abstract":"<p><p>The emergence of multidrug-resistant <i>Citrobacter freundii</i> poses a significant threat to public health. <i>C. freundii</i> isolates were collected from clinical patients in a Chinese hospital during 2020-2022. An unusual strain, GMU8049, was not susceptible to any of the antibiotics tested, including the novel β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam. Whole-genome sequencing (WGS) revealed that GMU8049 harbors a circular chromosome belonging to the rare ST257 and an IncX3 resistance plasmid. Genomic analysis revealed the coexistence of two β-lactamase genes, including plasmid-mediated <i>bla</i>_NDM-1 and chromosomal <i>bla</i>_CMY encoding a novel CMY variant, combined with an outer membrane porin deficiency, which may account for the extreme resistance to β-lactams. Conjugation experiment confirmed that the <i>bla</i>_NDM-1 resistance gene located on pGMU8049 could be successfully transferred to <i>Escherichia coli</i> EC600. The novel CMY variant had an amino acid substitution at position 106 (N106S) compared to the closely related CMY-51. Additionally, a GMU8049-specific truncation in an OmpK37 variant that produces a premature stop codon. Moreover, a variety of chromosome-located efflux pump coding genes and virulence-related genes were also identified. Analysis of strain GMU8049 in the context of other <i>C. freundii</i> strains reveals an open pan-genome and the presence of mobile genetic elements that can mediate horizontal gene transfer of antimicrobial resistance and virulence genes. Our work provides comprehensive insights into the genetic mechanisms of highly resistant <i>C. freundii</i>, highlighting the importance of genomic surveillance of this opportunistic pathogen as a high-risk population for emerging resistance and pathogenicity.IMPORTANCEEmerging pathogens exhibiting multi-, extremely, and pan-drug resistance are a major concern for hospitalized patients and the healthcare community due to limited antimicrobial treatment options and the potential for spread. Genomic technologies have enabled clinical surveillance of emerging pathogens and modeling of the evolution and transmission of antimicrobial resistance and virulence. Here, we report the genomic characterization of an extremely drug-resistant ST257 <i>Citrobacter freundii</i> clinical isolate. Genomic analysis of GMU8049 with a rare ST type and unusual phenotypes can provide information on how this extremely resistant clinical isolate has evolved, including the acquisition of <i>bla</i>_NDM-1 via the IncX3 plasmid and accumulation through chromosomal mutations leading to a novel CMY variant and deficiency of the outer membrane porin OmpK37. Our work highlights that the emergence of extremely resistant <i>C. freundii</i> poses a significant challenge to the treatment of clinical infections. Therefore, great efforts must be made to specifically monitor this opportunistic pathogen.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537026/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470088","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":"Comparison of the indirect immunofluorescence assay and a commercial ELISA to detect KSHV antibodies.","authors":"Valentin Leducq, Léa Ben Said, Sophie Sayon, Vincent Calvez, Anne-Geneviève Marcelin, Aude Jary","doi":"10.1128/spectrum.01186-24","DOIUrl":"10.1128/spectrum.01186-24","url":null,"abstract":"<p><p>Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus involved in several diseases. The gold standard for KSHV sero diagnosis remains the indirect immunofluorescence assay (IFA), which is time-consuming and operator-dependent. We compared this method with an enzyme-linked immunosorbent assay (ELISA) targeting solubilized KSHV whole-genome extract among positive (<i>n</i> = 49, including 76% of HIV-infected patients) and negative (<i>n</i> = 14) control groups. We also included 14 sera with equivocal IFA results. ELISA showed better performance in detecting KSHV antibodies (McNemar's test, <i>P =</i> 0.0455). The sensitivity and specificity of both methods were 79% (64-89) and 100% (66-100) for the IFA, respectively, and 94% (83-99) and 100% (66-100) for ELISA, respectively. All IFA equivocal results were either negative or positive with ELISA. ELISA is more reliable and could be a good alternative for determining KSHV serological status, particularly in the context of immunocompromised patients and equivocal serology with the IFA.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) sero status remains challenging because no perfect reference is available for the detection of KSHV antibodies. The current gold-standard method, the indirect immunofluorescence assay (IFA), has a very good specificity of close to 100%, but a lower sensitivity of around 80-85%, which decreases to 64-67% in immunocompromised patients. Additionally, this method is time-consuming and operator-dependent compared with new serological assays such as the enzyme-linked immunosorbent assay (ELISA). Thus, further research is still needed to improve KSHV sero diagnosis. Here, we compare the KSHV IgG ELISA kit assay (Advanced Biotechnologies Inc) with the gold-standard IFA, targeting the LANA-1 protein from latent BC-3 cell lines.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537116/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291334","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":"Heterogeneity of SARS-CoV-2 immune responses after the nationwide Omicron wave in China.","authors":"Jing Wu, Mingzheng Jiang, Jiwei Li, Xiaoyi Hu, Qiuyue Long, Shixu Song, Hongli Ye, Yukun He, Xinqian Ma, Wenyi Yu, Xi Chen, Lili Zhao, Fangfang Wu, Xiaoyong Chen, Jianshi Zheng, Minghui Wang, Binghan Zheng, Shuoqi Yang, Liang Bu, Qin Chen, Ke Li, Yali Zheng, Zhancheng Gao","doi":"10.1128/spectrum.01117-24","DOIUrl":"10.1128/spectrum.01117-24","url":null,"abstract":"<p><p>It remains unclear how previous infections and vaccinations influenced and shaped heterogeneous immune responses against Omicron and its variants in diverse populations in China. After the national wave of Omicron in early 2023, we evaluated serum levels of neutralizing antibodies (nAbs) against Omicron (B.1.1.529) and its variants (BA.5, BF.7, and CH1.1) in 33 COVID-19 convalescents and 40 uninfected vaccinees, using vesicular stomatitis virus-based pseudovirus neutralizing assay. In addition, we followed 34 Delta convalescent patients to compare their immune responses against Omicron before (late 2021) and after the Omicron wave (early 2023). NAbs at the acute phase of the disease were investigated in 50 Omicron inpatients, including 24 vaccinated and 26 unvaccinated patients. Among them, nasal mucosal IgA levels were measured in 42 subjects. Compared to vaccination, breakthrough infections significantly increased the breadth and magnitude of serum nAbs and mucosal IgA levels against Omicron variants. Exposure to Omicron but not Delta elicited stronger pan-Omicron responses. In Omicron inpatients, nAbs continued to rise as vaccination doses increased. However, in both vaccinees and convalescents, a fourth dose vaccination did not elicit higher nAbs against Omicron. Furthermore, nAbs against Omicron variants lasted longer than nAbs against WT SARS-CoV-2. Breakthrough infections of Omicron variants elicited specific immune responses against Omicron compared to vaccination and Delta infection. Although repeated vaccination revealed limited impacts on serum nAbs, populations at high risk of hospitalization may still benefit from continued vaccination.IMPORTANCEThe study described the specific humoral immunity against Omicron and its variants (BA.5, BF.7, and CH1.1) in diverse populations, including Delta-positive convalescent patients, Omicron-infected patients with a previous or current confirmed Delta infection, Omicron-positive patients, and healthy controls. In addition, we followed Delta convalescents for 1 year to evaluate the effect of a booster vaccine, breakthrough infection, and reinfection. Nasal mucosal IgA levels against SARS-CoV-2 were also examined. The findings of this study demonstrated the varied responses of individuals in different states following the outbreak of Omicron, highlighting the potential advantages of ongoing immunization for groups that are more vulnerable and have a greater likelihood of being hospitalized.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11536994/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291379","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":"Near-infrared <i>in vivo</i> imaging system for dynamic visualization of lung-colonizing bacteria in mouse pneumonia.","authors":"Daiki Yamaguchi, Go Kamoshida, Syun Kawakubo, Saki Azuma, Takamitsu Tsuji, Nobuo Kitada, Ryohei Saito-Moriya, Noriteru Yamada, Rentaro Tanaka, Ayane Okuda, Keisuke Ueyama, Shingo Isaka, Manaha Tomita, Ryuichi Nakano, Yuji Morita, Hisakazu Yano, Shojiro A Maki, Kinnosuke Yahiro, Shinichi Kato","doi":"10.1128/spectrum.00828-24","DOIUrl":"10.1128/spectrum.00828-24","url":null,"abstract":"<p><p><i>In vivo</i> imaging of bacterial infection models enables noninvasive and temporal analysis of individuals, enhancing our understanding of infectious disease pathogenesis. Conventional <i>in vivo</i> imaging methods for bacterial infection models involve the insertion of the bacterial luciferase LuxCDABE into the bacterial genome, followed by imaging using an expensive ultrasensitive charge-coupled device (CCD) camera. However, issues such as limited light penetration into the body and lack of versatility have been encountered. We focused on near-infrared (NIR) light, which penetrates the body effectively, and attempted to establish an <i>in vivo</i> imaging method to evaluate the number of lung-colonizing bacteria during the course of bacterial pneumonia. This was achieved by employing a novel versatile system that combines plasmid-expressing firefly luciferase bacteria, NIR substrate, and an inexpensive, scientific complementary metal-oxide semiconductor (sCMOS) camera. The D-luciferin derivative \"TokeOni,\" capable of emitting NIR bioluminescence, was utilized in a mouse lung infection model of <i>Acinetobacter baumannii</i>, an opportunistic pathogen that causes pneumonia and is a concern due to drug resistance. TokeOni exhibited the highest sensitivity in detecting bacteria colonizing the mouse lungs compared with other detection systems such as LuxCDABE, enabling the monitoring of changes in bacterial numbers over time and the assessment of antimicrobial agent efficacy. Additionally, it was effective in detecting <i>A. baumannii</i> clinical isolates and <i>Klebsiella pneumoniae</i>. The results of this study are expected to be used in the analysis of animal models of infectious diseases for assessing the efficacy of therapeutic agents and understanding disease pathogenesis.</p><p><strong>Importance: </strong>Conventional animal models of infectious diseases have traditionally relied upon average assessments involving numerous individuals, meaning they do not directly reflect changes in the pathology of an individual. Moreover, in recent years, ethical concerns have resulted in the demand to reduce the number of animals used in such models. Although <i>in vivo</i> imaging offers an effective approach for longitudinally evaluating the pathogenesis of infectious diseases in individual animals, a standardized method has not yet been established. To our knowledge, this study is the first to develop a highly versatile <i>in vivo</i> pulmonary bacterial quantification system utilizing near-infrared luminescence, plasmid-mediated expression of firefly luciferase in bacteria, and a scientific complementary metal-oxide semiconductor camera. Our research holds promise as a useful tool for assessing the efficacy of therapeutic drugs and pathogenesis of infectious diseases.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537041/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291386","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}