Pub Date : 2024-09-14DOI: 10.1101/2024.09.13.612945
Van N Trinh, Nisha J Mulakken, Kara L Nelson, Nicholas A Be, Rose S Kantor
Quantitative polymerase chain reaction (qPCR) assays are sensitive molecular tools commonly used to quantify pathogens in environmental samples. These assays have become a staple of wastewater-based surveillance and often form the basis of quantitative microbial risk assessments. However, PCR assays may fail to capture all of their intended targets due to signature erosion over time. Here, we performed an in silico analysis of four human enterovirus PCR assays to assess signature erosion against the NCBI virus database. The predicted number of genomes hit by each assay rose alongside total genomes in the database through 2010 but the proportion of predicted hits began to level off with the emergence of the clinically important enterovirus D-68. We found that although all assays captured a majority of enterovirus species, only one recently developed assay adequately captured enterovirus D species. Some assays also appeared more likely to capture non-human enteroviruses than others, an important consideration for data interpretation. We conclude that broad-spectrum virus assays applied to environmental samples should be regularly checked against expanding sequence databases and provide methods to do so.
{"title":"In silico analysis reveals differential targeting of enterovirus species by commonly used PCR assays","authors":"Van N Trinh, Nisha J Mulakken, Kara L Nelson, Nicholas A Be, Rose S Kantor","doi":"10.1101/2024.09.13.612945","DOIUrl":"https://doi.org/10.1101/2024.09.13.612945","url":null,"abstract":"Quantitative polymerase chain reaction (qPCR) assays are sensitive molecular tools commonly used to quantify pathogens in environmental samples. These assays have become a staple of wastewater-based surveillance and often form the basis of quantitative microbial risk assessments. However, PCR assays may fail to capture all of their intended targets due to signature erosion over time. Here, we performed an in silico analysis of four human enterovirus PCR assays to assess signature erosion against the NCBI virus database. The predicted number of genomes hit by each assay rose alongside total genomes in the database through 2010 but the proportion of predicted hits began to level off with the emergence of the clinically important enterovirus D-68. We found that although all assays captured a majority of enterovirus species, only one recently developed assay adequately captured enterovirus D species. Some assays also appeared more likely to capture non-human enteroviruses than others, an important consideration for data interpretation. We conclude that broad-spectrum virus assays applied to environmental samples should be regularly checked against expanding sequence databases and provide methods to do so.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bacteria in polymicrobial habitats are constantly exposed to biotic threats from bacteriophages, antagonistic bacteria, and predatory eukaryotes. These antagonistic interactions play crucial roles in shaping the evolution and physiology of bacteria. To survive, bacteria have evolved mechanisms to protect themselves from such attacks, but the fitness costs of resisting one threat and rendering bacteria susceptible to others remain unappreciated. Here, we examined the fitness consequences of bacteriophage resistance in Salmonella enterica, revealing that phage-resistant variants exhibited significant fitness loss upon co-culture with competitor bacteria. These phage-resistant strains display varying degrees of lipopolysaccharide (LPS) deficiency and increased susceptibility to contact-dependent interbacterial antagonism, such as the type VI secretion system (T6SS). Utilizing mutational analyses and atomic force microscopy, we show that the long-modal length O-antigen of LPS serves as a protective barrier against T6SS-mediated intoxication. Notably, this competitive disadvantage can also be triggered independently by phages possessing LPS-targeting endoglycosidase in their tail spike proteins, which actively cleave the O-antigen upon infection. Our findings reveal two distinct mechanisms of phage-mediated LPS modifications that modulate interbacterial competition, shedding light on the dynamic microbial interplay within mixed populations.
多微生物栖息地中的细菌经常面临来自噬菌体、拮抗细菌和捕食性真核生物的生物威胁。这些拮抗作用对细菌的进化和生理起着至关重要的作用。为了生存，细菌进化出了保护自身免受此类攻击的机制，但抵御一种威胁并使细菌易受其他威胁影响的健康成本仍未得到重视。在这里，我们研究了肠炎沙门氏菌对噬菌体抗性的适应性后果，发现抗噬菌体变异株在与竞争细菌共培养时表现出显著的适应性损失。这些噬菌体抗性菌株表现出不同程度的脂多糖（LPS）缺乏症，并且对依赖接触的细菌间拮抗作用（如 VI 型分泌系统（T6SS））的敏感性增加。利用突变分析和原子力显微镜，我们发现 LPS 的长模态长度 O 抗原是抵御 T6SS 介导的中毒的保护屏障。值得注意的是，噬菌体的尾尖蛋白中具有 LPS 靶向内糖苷酶，在感染时会主动裂解 O 抗原，这也能单独触发这种竞争劣势。我们的研究结果揭示了噬菌体介导的 LPS 修饰调节细菌间竞争的两种不同机制，从而揭示了混合种群中微生物之间的动态相互作用。
{"title":"Surface-mediated Bacteriophage Defense Incurs Fitness Tradeoffs for Interbacterial Antagonism","authors":"Chia-En Tsai, Feng-Qi Wang, Chih-Wen Yang, Ling-Li Yang, Yung-Chih Chen, Po-Yin Chen, Ing-Shouh Hwang, See-Yeun Ting","doi":"10.1101/2024.09.13.612980","DOIUrl":"https://doi.org/10.1101/2024.09.13.612980","url":null,"abstract":"Bacteria in polymicrobial habitats are constantly exposed to biotic threats from bacteriophages, antagonistic bacteria, and predatory eukaryotes. These antagonistic interactions play crucial roles in shaping the evolution and physiology of bacteria. To survive, bacteria have evolved mechanisms to protect themselves from such attacks, but the fitness costs of resisting one threat and rendering bacteria susceptible to others remain unappreciated. Here, we examined the fitness consequences of bacteriophage resistance in Salmonella enterica, revealing that phage-resistant variants exhibited significant fitness loss upon co-culture with competitor bacteria. These phage-resistant strains display varying degrees of lipopolysaccharide (LPS) deficiency and increased susceptibility to contact-dependent interbacterial antagonism, such as the type VI secretion system (T6SS). Utilizing mutational analyses and atomic force microscopy, we show that the long-modal length O-antigen of LPS serves as a protective barrier against T6SS-mediated intoxication. Notably, this competitive disadvantage can also be triggered independently by phages possessing LPS-targeting endoglycosidase in their tail spike proteins, which actively cleave the O-antigen upon infection. Our findings reveal two distinct mechanisms of phage-mediated LPS modifications that modulate interbacterial competition, shedding light on the dynamic microbial interplay within mixed populations.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259895","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1101/2024.09.13.612987
Mi Ae Park, Sharifah Nora Ahmad Almunawar, Rachel Rui Xia Lim, Sumanto Haldar, Christiani Jeyakumar Henry, Oleg V Moskvin
Clostridium leptum, a key player in gut butyrate production, has a profound impact on various facets of intestinal health. A recent clinical trial highlighted a significant increase in the relative abundance of this species in response to dietary interventions using beneficial oils. We isolated microbial strains corresponding to 'Clostridium leptum' (at the 16S rRNA gene similarity level) and sequenced their genomes. All three genomes were successfully reconstructed, maintaining the chromosome as a single contig. Subsequent genome-wide analysis unveiled the phylogenetic diversity of the isolates, including the discovery of a new species - Gallacutalibacter singaporense. Based on the reconstructed metabolic model, we predicted growth condition patterns of this new species and confirmed the predictions in vitro. Leveraging the assembled genomes, we dissected the components of the strong dietary intervention response signal previously ascribed to 'C.leptum' and revealed distinct individual dynamics of all three bacteria in the clinical trial context. The transitional behavior of the novel species, in particular, revealed intriguing patterns, blazing the path to uncovering previously unrecognized interactions along the diet - gut microbiome - human health axis.
{"title":"Genomic Reconstruction and Dietary Response Assessment of Three Acutalibacteraceae Bacteria Isolated from Fecal Samples of Singapore Subjects.","authors":"Mi Ae Park, Sharifah Nora Ahmad Almunawar, Rachel Rui Xia Lim, Sumanto Haldar, Christiani Jeyakumar Henry, Oleg V Moskvin","doi":"10.1101/2024.09.13.612987","DOIUrl":"https://doi.org/10.1101/2024.09.13.612987","url":null,"abstract":"Clostridium leptum, a key player in gut butyrate production, has a profound impact on various facets of intestinal health. A recent clinical trial highlighted a significant increase in the relative abundance of this species in response to dietary interventions using beneficial oils. We isolated microbial strains corresponding to 'Clostridium leptum' (at the 16S rRNA gene similarity level) and sequenced their genomes. All three genomes were successfully reconstructed, maintaining the chromosome as a single contig. Subsequent genome-wide analysis unveiled the phylogenetic diversity of the isolates, including the discovery of a new species - Gallacutalibacter singaporense. Based on the reconstructed metabolic model, we predicted growth condition patterns of this new species and confirmed the predictions in vitro. Leveraging the assembled genomes, we dissected the components of the strong dietary intervention response signal previously ascribed to 'C.leptum' and revealed distinct individual dynamics of all three bacteria in the clinical trial context. The transitional behavior of the novel species, in particular, revealed intriguing patterns, blazing the path to uncovering previously unrecognized interactions along the diet - gut microbiome - human health axis.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259896","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Papain-like protease (PLpro) plays a critical role in both viral polyprotein processing and host antiviral immune suppression in SARS-CoV-2 infection, which causes COVID-19. Although several drugs have been approved for COVID-19, such as Remdesivir, Nirmatrelvir, etc., none of the PLpro inhibitors have been approved for the treatment of COVID-19. The advent of artificial intelligence-based drug design methods has significantly accelerated the process of drug discovery. In current study, by harnessing the power of a topology constrained molecular generative model, we discovered a novel series of PLpro inhibitors with strong potency against prevalent SARS-CoV-2 variants. Following a structure based computational approach for optimization, our lead compound, GZNL-2002, achieved decent PLpro inhibitory potency and favorable pharmacokinetic properties, which warrants further development as a potential candidate compound for COVID-19 disease.
{"title":"Discovery of novel quinoline papain-like protease inhibitors for COVID-19 through topology constrained molecular generative model","authors":"Jinsai Shang, Ting Ran, Yongzhi Lu, Qi Yang, Guihua Zhang, Peiqi Zhou, Wenqi Li, Minyuan Xu, Jielin Tang, Minxian Dai, Jinpeng Zhong, Hua Chen, Pan He, Anqi Zhou, Bao Xue, Jiayi Chen, Jiyun Zhang, Kunzhong Wu, Xinyu Wu, Miru Tang, Xinwen Chen, Hongming Chen","doi":"10.1101/2024.09.07.611841","DOIUrl":"https://doi.org/10.1101/2024.09.07.611841","url":null,"abstract":"Papain-like protease (PL<sup>pro</sup>) plays a critical role in both viral polyprotein processing and host antiviral immune suppression in SARS-CoV-2 infection, which causes COVID-19. Although several drugs have been approved for COVID-19, such as Remdesivir, Nirmatrelvir, etc., none of the PL<sup>pro</sup> inhibitors have been approved for the treatment of COVID-19. The advent of artificial intelligence-based drug design methods has significantly accelerated the process of drug discovery. In current study, by harnessing the power of a topology constrained molecular generative model, we discovered a novel series of PL<sup>pro</sup> inhibitors with strong potency against prevalent SARS-CoV-2 variants. Following a structure based computational approach for optimization, our lead compound, GZNL-2002, achieved decent PL<sup>pro</sup> inhibitory potency and favorable pharmacokinetic properties, which warrants further development as a potential candidate compound for COVID-19 disease.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213987","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Background: Preterm birth (birth before 37 weeks of gestational age) is a frequent and severe adverse pregnancy outcome. Despite the growing number of scientific studies highlighting the link between vaginal microbiota composition and adverse pregnancy outcomes, data remain controversial. Objectives: To identify microbiota signatures of preterm labor and premature rupture of outer membranes; to determine microbiological risk factors for preterm birth. Study Design: We conducted an observational, prospective, longitudinal cohort study from August 2018 until June 2023 in 3 maternity wards from university hospitals in the Paris, France, area. Women were categorized in 4 groups including a control group, and 3 groups of increasing risk of intrauterine infection and preterm birth: prelabor rupture of membranes at term, preterm labor, and preterm premature rupture of membranes. Demographic, clinical data, past medical and obstetrical history and pregnancy outcome were collected. Vaginal swabs were collected at admission and were analyzed using culturomics. The association between bacterial species and the cohort groups and eventually preterm birth was studied in univariate analyses. Adjusted odds ratio (aOR) and 95% confidence intervals (95% CI) were calculated in multivariable analyses. Results: A total of 2,476 women were included, of whom 1,068 (43.1%) in the control group, 477 (19.3%) with term premature rupture of outer membranes, 495 (20.0%) with preterm labor, and 436 (17.6%) with preterm premature rupture of outer membranes. Together with demographic features such as ethnicity and obstetrical history, several vaginal microbiota signatures were identified as correlated with pregnancy outcome. In multivariable analysis, prelabor rupture of membranes at term was associated with enterobacteria (aOR 1.97, 95% CI 1.46-2.65) and Gardnerella vaginalis (aOR 5.19, 95% CI 2.22-13.78); preterm labor with lactobacilli depletion (aOR 1.49, 95% CI 1.08-2.06), enterobacteria (aOR 1.86, 95% CI 1.36-2.53) and G. vaginalis (aOR 4.62, 95% CI 1.86-13.34); preterm premature rupture of membranes with lactobacilli depletion (aOR 2.04, 95% CI 1.49-2.80) and enterobacteria (aOR 2.38, 95% CI 1.74-3.24). Finally, together with gestational diabetes, lactobacilli depletion and enterobacteria both represented risk factors for preterm birth, especially in singleton pregnancies (aOR 1.54, 95% CI 1.05-2.28 and aOR 1.62, 95% CI 1.11-2.36, respectively). Conclusions: In this large cohort study, we identified on the one hand, G. vaginalis as associated with prelabor rupture of membranes at term and preterm labor, and on the other hand, lactobacilli depletion and enterobacteria as risk factors for preterm labor, preterm premature rupture of outer membranes, and preterm birth, emphasizing the importance of a healthy microbiota in pregnancy outcome. Further studies are needed to address the benefits of adapted antibiotic prophylaxes and probiotics aiming at restoring a healthy microb
背景：早产（胎龄 37 周前出生）是一种常见的严重不良妊娠结局。尽管越来越多的科学研究强调了阴道微生物群组成与不良妊娠结局之间的联系，但数据仍存在争议：确定早产和胎膜早破的微生物群特征；确定早产的微生物风险因素。研究设计：我们从 2018 年 8 月至 2023 年 6 月在法国巴黎地区 3 所大学医院的产科病房开展了一项观察性、前瞻性、纵向队列研究。妇女被分为 4 组，包括对照组，以及宫内感染和早产风险增加的 3 组：临产前胎膜破裂、早产和早产胎膜早破。收集了人口统计学、临床数据、既往病史和产科病史以及妊娠结果。入院时采集了阴道拭子，并使用培养组学进行了分析。在单变量分析中研究了细菌种类与队列分组和最终早产之间的关系。在多变量分析中计算了调整后的几率比（aOR）和 95% 置信区间（95% CI）。结果共纳入 2,476 名妇女，其中对照组 1,068 人（43.1%），477 人（19.3%）为足月胎膜早破，495 人（20.0%）为早产，436 人（17.6%）为早产胎膜早破。与种族和产科病史等人口统计学特征一起，确定了与妊娠结局相关的几种阴道微生物群特征。在多变量分析中，临产时胎膜早破与肠杆菌（aOR 1.97，95% CI 1.46-2.65）和阴道加德纳菌（aOR 5.19，95% CI 2.22-13.78）有关；早产与乳酸杆菌耗竭（aOR 1.49，95% CI 1.08-2.06）、肠杆菌（aOR 1.86，95% CI 1.36-2.53）和阴道杆菌（aOR 4.62，95% CI 1.86-13.34）；早产胎膜早破与乳酸杆菌耗竭（aOR 2.04，95% CI 1.49-2.80）和肠杆菌（aOR 2.38，95% CI 1.74-3.24）有关。最后，乳酸菌耗竭和肠道细菌与妊娠糖尿病都是早产的风险因素，尤其是在单胎妊娠中（aOR 1.54，95% CI 1.05-2.28；aOR 1.62，95% CI 1.11-2.36）：在这项大型队列研究中，我们一方面发现阴道杆菌与临产前胎膜破裂和早产有关，另一方面发现乳酸杆菌耗竭和肠杆菌是早产、胎膜早破和早产的风险因素，这强调了健康的微生物群对妊娠结局的重要性。还需要进一步的研究来探讨旨在恢复健康微生物群的抗生素预防和益生菌对降低早产风险的益处。
{"title":"Vaginal microbiota as a predictor of preterm birth: an observational cohort study","authors":"Laura Lesimple, Jessica Rousseau, Celine Plainvert, Luce Landraud, Nathalie Grall, Francois Goffinet, Pierre-Yves Ancel, Christophe Pannetier, Claire Poyart, Laurent Mandelbrot, Asmaa Tazi","doi":"10.1101/2024.09.11.612423","DOIUrl":"https://doi.org/10.1101/2024.09.11.612423","url":null,"abstract":"Background: Preterm birth (birth before 37 weeks of gestational age) is a frequent and severe adverse pregnancy outcome. Despite the growing number of scientific studies highlighting the link between vaginal microbiota composition and adverse pregnancy outcomes, data remain controversial.\u0000Objectives: To identify microbiota signatures of preterm labor and premature rupture of outer membranes; to determine microbiological risk factors for preterm birth. Study Design: We conducted an observational, prospective, longitudinal cohort study from August 2018 until June 2023 in 3 maternity wards from university hospitals in the Paris, France, area. Women were categorized in 4 groups including a control group, and 3 groups of increasing risk of intrauterine infection and preterm birth: prelabor rupture of membranes at term, preterm labor, and preterm premature rupture of membranes. Demographic, clinical data, past medical and obstetrical history and pregnancy outcome were collected. Vaginal swabs were collected at admission and were analyzed using culturomics. The association between bacterial species and the cohort groups and eventually preterm birth was studied in univariate analyses. Adjusted odds ratio (aOR) and 95% confidence intervals (95% CI) were calculated in multivariable analyses. Results: A total of 2,476 women were included, of whom 1,068 (43.1%) in the control group, 477 (19.3%) with term premature rupture of outer membranes, 495 (20.0%) with preterm labor, and 436 (17.6%) with preterm premature rupture of outer membranes. Together with demographic features such as ethnicity and obstetrical history, several vaginal microbiota signatures were identified as correlated with pregnancy outcome. In multivariable analysis, prelabor rupture of membranes at term was associated with enterobacteria (aOR 1.97, 95% CI 1.46-2.65) and Gardnerella vaginalis (aOR 5.19, 95% CI 2.22-13.78); preterm labor with lactobacilli depletion (aOR 1.49, 95% CI 1.08-2.06), enterobacteria (aOR 1.86, 95% CI 1.36-2.53) and G. vaginalis (aOR 4.62, 95% CI 1.86-13.34); preterm premature rupture of membranes with lactobacilli depletion (aOR 2.04, 95% CI 1.49-2.80) and enterobacteria (aOR 2.38, 95% CI 1.74-3.24). Finally, together with gestational diabetes, lactobacilli depletion and enterobacteria both represented risk factors for preterm birth, especially in singleton pregnancies (aOR 1.54, 95% CI 1.05-2.28 and aOR 1.62, 95% CI 1.11-2.36, respectively).\u0000Conclusions: In this large cohort study, we identified on the one hand, G. vaginalis as associated with prelabor rupture of membranes at term and preterm labor, and on the other hand, lactobacilli depletion and enterobacteria as risk factors for preterm labor, preterm premature rupture of outer membranes, and preterm birth, emphasizing the importance of a healthy microbiota in pregnancy outcome. Further studies are needed to address the benefits of adapted antibiotic prophylaxes and probiotics aiming at restoring a healthy microb","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213986","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.10.612295
Dhinesh Periyasamy, Yanyun Huang, Janet E Hill
Bovine reproductive failure, which includes infertility, abortion, and stillbirth in cattle, leads to significant economic losses for beef and milk producers. Diagnosing the infectious causes of bovine reproductive failure is challenging as there are multiple pathogens associated with it. The traditional stepwise approach to diagnostic testing is time-consuming and can cause significant delays. In this study, we have developed a syndromic next-generation sequencing panel (BovReproSeq), for the simultaneous detection of 17 pathogens associated with bovine reproductive failure. This targeted approach involves amplifying multiple pathogen-specific targets using ultra-multiplex PCR, followed by sequencing with the Oxford Nanopore platform and subsequent analysis of the data using a custom bioinformatic pipeline to determine the presence or absence of pathogens. We tested 116 clinical samples and found that BovReproSeq results matched with current diagnostic methods for 93% of the samples, and most of the disagreements occurring in samples with very low pathogen loads (Ct > 35). At the optimal read-count threshold of 10 reads (minimum number of reads to classify the sample as positive), the sensitivity of the assay was approximately 82%, while specificity was 100%. The overall accuracy of the assay was 98.8%. Matthew's Correlation Coefficient was approximately 0.90 and F1 score (harmonic mean of Precision and Recall) was 0.90, indicating excellent overall performance. Our study presents a significant advancement in detecting the infectious agents associated with bovine reproductive failure and the BovReproSeq panel's ability to detect 17 pathogens makes it a promising tool for veterinary diagnostics.
{"title":"Targeted syndromic next-generation sequencing panel for simultaneous detection of pathogens associated with bovine reproductive failure","authors":"Dhinesh Periyasamy, Yanyun Huang, Janet E Hill","doi":"10.1101/2024.09.10.612295","DOIUrl":"https://doi.org/10.1101/2024.09.10.612295","url":null,"abstract":"Bovine reproductive failure, which includes infertility, abortion, and stillbirth in cattle, leads to significant economic losses for beef and milk producers. Diagnosing the infectious causes of bovine reproductive failure is challenging as there are multiple pathogens associated with it. The traditional stepwise approach to diagnostic testing is time-consuming and can cause significant delays. In this study, we have developed a syndromic next-generation sequencing panel (BovReproSeq), for the simultaneous detection of 17 pathogens associated with bovine reproductive failure. This targeted approach involves amplifying multiple pathogen-specific targets using ultra-multiplex PCR, followed by sequencing with the Oxford Nanopore platform and subsequent analysis of the data using a custom bioinformatic pipeline to determine the presence or absence of pathogens. We tested 116 clinical samples and found that BovReproSeq results matched with current diagnostic methods for 93% of the samples, and most of the disagreements occurring in samples with very low pathogen loads (Ct > 35). At the optimal read-count threshold of 10 reads (minimum number of reads to classify the sample as positive), the sensitivity of the assay was approximately 82%, while specificity was 100%. The overall accuracy of the assay was 98.8%. Matthew's Correlation Coefficient was approximately 0.90 and F1 score (harmonic mean of Precision and Recall) was 0.90, indicating excellent overall performance. Our study presents a significant advancement in detecting the infectious agents associated with bovine reproductive failure and the BovReproSeq panel's ability to detect 17 pathogens makes it a promising tool for veterinary diagnostics.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.11.612452
Mario Kapitan, Maria Joanna Niemiec, Nicolas Millet, Philipp Brandt, Md Estiak Khan Chowdhury, Anna Czapka, Ketema Abdissa, Franziska Hoffmann, Anna Lange, Mark Veleba, Sandor Nietzsche, Alexander Sandy Mosig, Bettina Loffler, Mike Marquet, Oliwia Makarewicz, Kimberly A. Kline, Slavena Vylkova, Marc Swidergall, Ilse D. Jacobsen
The fungus Candida albicans and the Gram-positive bacterium Enterococcus faecalis share mucosal niches in the human body. As opportunistic pathogens, both are found to expand population size during dysbiosis, and can cause severe systemic infections in susceptible individuals. Here, we show that the presence of C. albicans results in increased host cell damage by E. faecalis. Furthermore, E. faecalis aggravates oropharyngeal candidiasis in mice. Increased damage is mediated by enterococcal cytolysin, and involves both physical interaction and altered glucose availability. Physical interaction promotes accumulation of bacteria on host cells, facilitating contact of cytolysin with host cells. Glucose depletion by the metabolic activity of the fungus sensitized host cells to cytolysin. This work illustrates how a complex interplay between fungi and bacteria can result in detrimental consequences for the host.
{"title":"Synergistic cross-kingdom host cell damage between Candida albicans and Enterococcus faecalis","authors":"Mario Kapitan, Maria Joanna Niemiec, Nicolas Millet, Philipp Brandt, Md Estiak Khan Chowdhury, Anna Czapka, Ketema Abdissa, Franziska Hoffmann, Anna Lange, Mark Veleba, Sandor Nietzsche, Alexander Sandy Mosig, Bettina Loffler, Mike Marquet, Oliwia Makarewicz, Kimberly A. Kline, Slavena Vylkova, Marc Swidergall, Ilse D. Jacobsen","doi":"10.1101/2024.09.11.612452","DOIUrl":"https://doi.org/10.1101/2024.09.11.612452","url":null,"abstract":"The fungus Candida albicans and the Gram-positive bacterium Enterococcus faecalis share mucosal niches in the human body. As opportunistic pathogens, both are found to expand population size during dysbiosis, and can cause severe systemic infections in susceptible individuals. Here, we show that the presence of C. albicans results in increased host cell damage by E. faecalis. Furthermore, E. faecalis aggravates oropharyngeal candidiasis in mice. Increased damage is mediated by enterococcal cytolysin, and involves both physical interaction and altered glucose availability. Physical interaction promotes accumulation of bacteria on host cells, facilitating contact of cytolysin with host cells. Glucose depletion by the metabolic activity of the fungus sensitized host cells to cytolysin. This work illustrates how a complex interplay between fungi and bacteria can result in detrimental consequences for the host.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213985","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1101/2024.09.12.612520
Kimberly A Dowd, Michelle Schroeder, Egan Sanchez, Beniah Brumbaugh, Bryant M Foreman, Katherine E Burgomaster, Wei Shi, Lingshu Wang, Natalie Caputo, David Gordon, Cindi L Schwartz, Bryan T Hansen, Maya Aleshnick, Wing-Pui Kong, Kaitlyn M Morabito, Heather D Hickman, Barney S Graham, Elizabeth R Fischer, Theodore C Pierson
Flavivirus assembly at the endoplasmic reticulum is driven by the structural proteins envelope (E) and premembrane (prM). Here, contrary to the established paradigm for flavivirus assembly, we demonstrate that the biogenesis of flavivirus particles does not require an intact prM nor proteolytic activation. The expression of E preceded by a truncated version of prM (M-E) was sufficient for the formation of non-infectious Zika virus subviral particles and pseudo-infectious reporter virions. Subviral particles encoded by a ZIKV M-E DNA vaccine elicited a neutralizing antibody response that was insensitive to the virion maturation state, a feature of flavivirus humoral immunity shown to correlate with protection. M-E vaccines that uniformly present structural features shared with mature virions offer a higher quality and broadly applicable approach to flavivirus vaccination.
黄病毒在内质网的组装是由包膜（E）和膜前（prM）结构蛋白驱动的。在这里，与黄病毒组装的既定模式相反，我们证明了黄病毒颗粒的生物生成不需要完整的prM，也不需要蛋白水解激活。在表达 E 之前先表达截短版 prM（M-E）足以形成非感染性寨卡病毒亚病毒粒子和假感染性报告病毒。由 ZIKV M-E DNA 疫苗编码的亚病毒颗粒可引起对病毒成熟状态不敏感的中和抗体反应，而黄病毒体液免疫的这一特征已被证明与保护相关。与成熟病毒具有相同结构特征的M-E疫苗为黄病毒疫苗接种提供了一种质量更高、适用范围更广的方法。
{"title":"pr-independent biogenesis of infectious mature Zika virus particles","authors":"Kimberly A Dowd, Michelle Schroeder, Egan Sanchez, Beniah Brumbaugh, Bryant M Foreman, Katherine E Burgomaster, Wei Shi, Lingshu Wang, Natalie Caputo, David Gordon, Cindi L Schwartz, Bryan T Hansen, Maya Aleshnick, Wing-Pui Kong, Kaitlyn M Morabito, Heather D Hickman, Barney S Graham, Elizabeth R Fischer, Theodore C Pierson","doi":"10.1101/2024.09.12.612520","DOIUrl":"https://doi.org/10.1101/2024.09.12.612520","url":null,"abstract":"Flavivirus assembly at the endoplasmic reticulum is driven by the structural proteins envelope (E) and premembrane (prM). Here, contrary to the established paradigm for flavivirus assembly, we demonstrate that the biogenesis of flavivirus particles does not require an intact prM nor proteolytic activation. The expression of E preceded by a truncated version of prM (M-E) was sufficient for the formation of non-infectious Zika virus subviral particles and pseudo-infectious reporter virions. Subviral particles encoded by a ZIKV M-E DNA vaccine elicited a neutralizing antibody response that was insensitive to the virion maturation state, a feature of flavivirus humoral immunity shown to correlate with protection. M-E vaccines that uniformly present structural features shared with mature virions offer a higher quality and broadly applicable approach to flavivirus vaccination.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213988","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1101/2024.09.12.612745
Erica R Fuhrmeister, Sooyeol Kim, Shruteek A Mairal, Caroline McCormack, Benard Chieng, Jenna M Swarthout, Abigail Harvey Paulos, Sammy M Njenga, Amy J Pickering
Antimicrobial resistance (AMR) aligns with a One Health framework in that resistant bacteria and antibiotic resistance genes (ARGs) can be transmitted between humans, animals, and the environment. However, there is a critical need to more precisely understand how and to what extent AMR is exchanged between animals and humans. Metagenomic sequencing has low detection for rare targets such as ARGs, while whole genome sequencing of isolates is burdensome and misses exchange between uncultured bacterial species. We developed a novel, targeted sequencing assay using CRISPR-Cas9 to selectively sequence ARGs and their genomic context with long-read sequencing. Using this method, termed Context-Seq, we investigated overlapping AMR elements containing the ARGs blaCTX-M and blaTEM between adults, children, poultry, and dogs in animal-owning households in Nairobi, Kenya. We identified 22 genetically distinct clusters (> 80%ID over ≥ 3000 bp) containing blaTEM and one cluster containing blaCTX-M that were shared within and between households. Half of the clusters were shared between humans and animals, while the other half were shared only between animals (poultry-poultry, dog-dog, and dog-poultry). We identified potentially pathogenic hosts of ARGs including Escherichia coli, Klebsiella pneumonia, and Haemophilus influenzae across sample types. Context-Seq complements conventional methods to obtain an additional view of bacterial and mammalian hosts in the proliferation of AMR.
{"title":"Context-Seq: CRISPR-Cas9 Targeted Nanopore Sequencing for Transmission Dynamics of Antimicrobial Resistance","authors":"Erica R Fuhrmeister, Sooyeol Kim, Shruteek A Mairal, Caroline McCormack, Benard Chieng, Jenna M Swarthout, Abigail Harvey Paulos, Sammy M Njenga, Amy J Pickering","doi":"10.1101/2024.09.12.612745","DOIUrl":"https://doi.org/10.1101/2024.09.12.612745","url":null,"abstract":"Antimicrobial resistance (AMR) aligns with a One Health framework in that resistant bacteria and antibiotic resistance genes (ARGs) can be transmitted between humans, animals, and the environment. However, there is a critical need to more precisely understand how and to what extent AMR is exchanged between animals and humans. Metagenomic sequencing has low detection for rare targets such as ARGs, while whole genome sequencing of isolates is burdensome and misses exchange between uncultured bacterial species. We developed a novel, targeted sequencing assay using CRISPR-Cas9 to selectively sequence ARGs and their genomic context with long-read sequencing. Using this method, termed Context-Seq, we investigated overlapping AMR elements containing the ARGs blaCTX-M and blaTEM between adults, children, poultry, and dogs in animal-owning households in Nairobi, Kenya. We identified 22 genetically distinct clusters (> 80%ID over ≥ 3000 bp) containing blaTEM and one cluster containing blaCTX-M that were shared within and between households. Half of the clusters were shared between humans and animals, while the other half were shared only between animals (poultry-poultry, dog-dog, and dog-poultry). We identified potentially pathogenic hosts of ARGs including Escherichia coli, Klebsiella pneumonia, and Haemophilus influenzae across sample types. Context-Seq complements conventional methods to obtain an additional view of bacterial and mammalian hosts in the proliferation of AMR.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1101/2024.09.11.612487
Skyler Granatir, Francisco M. Acosta, Christina Pantoja, Johann Tailor, Angus Fuori, Bill Dower, Henry Marr, Peter W Ramirez
Infection and transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to pose a global public health concern. Using electromagnetic waves represents an alternative strategy to inactivate pathogenic viruses such as SARS-CoV-2 and reduce overall transmission. However, whether electromagnetic waves reduce SARS-CoV-2 infectivity is unclear. Here, we adapted a coplanar waveguide (CPW) to identify electromagnetic waves that could neutralize SARS-CoV-2 virus-like particles (SC2-VLPs). Treatment of SC2-VLPs, particularly at frequencies between 2.5-3.5 GHz at an electric field of 400 V/m for 2 minutes, reduced infectivity. Exposure to a frequency of 3.1 GHz decreased the binding of SC2-VLPs to antibodies directed against the Spike S1 subunit receptor binding domain (RBD). These results suggest that electromagnetic waves alter the conformation of Spike, thereby reducing viral attachment to host cell receptors. Overall, this data provides proof-of-concept in using electromagnetic waves for sanitation and prevention efforts to curb the transmission of SARS-CoV-2 and potentially other pathogenic enveloped viruses.
{"title":"Electromagnetic waves destabilize the SARS-CoV-2 Spike protein and reduce SARS-CoV-2 Virus-Like Particle (SC2-VLP) infectivity","authors":"Skyler Granatir, Francisco M. Acosta, Christina Pantoja, Johann Tailor, Angus Fuori, Bill Dower, Henry Marr, Peter W Ramirez","doi":"10.1101/2024.09.11.612487","DOIUrl":"https://doi.org/10.1101/2024.09.11.612487","url":null,"abstract":"Infection and transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to pose a global public health concern. Using electromagnetic waves represents an alternative strategy to inactivate pathogenic viruses such as SARS-CoV-2 and reduce overall transmission. However, whether electromagnetic waves reduce SARS-CoV-2 infectivity is unclear. Here, we adapted a coplanar waveguide (CPW) to identify electromagnetic waves that could neutralize SARS-CoV-2 virus-like particles (SC2-VLPs). Treatment of SC2-VLPs, particularly at frequencies between 2.5-3.5 GHz at an electric field of 400 V/m for 2 minutes, reduced infectivity. Exposure to a frequency of 3.1 GHz decreased the binding of SC2-VLPs to antibodies directed against the Spike S1 subunit receptor binding domain (RBD). These results suggest that electromagnetic waves alter the conformation of Spike, thereby reducing viral attachment to host cell receptors. Overall, this data provides proof-of-concept in using electromagnetic waves for sanitation and prevention efforts to curb the transmission of SARS-CoV-2 and potentially other pathogenic enveloped viruses.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214019","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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