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Activity-based protein profiling of serine hydrolases and penicillin-binding proteins in Enterococcus faecium 基于活性的粪肠球菌丝氨酸水解酶和青霉素结合蛋白特征分析
Pub Date : 2024-05-15 DOI: 10.1093/femsmc/xtae015
J. S. Grunnvåg, K. Hegstad, Christian S. Lentz
Enterococcus faecium is a gut commensal bacterium that is gaining increasing relevance as an opportunistic, nosocomial pathogen. Its high level of intrinsic and acquired antimicrobial resistance is causing a lack of treatment options, particularly for infections with vancomycin-resistant strains, and prioritizes the identification and functional validation of novel druggable targets. Here, we use activity-based protein profiling (ABPP), a chemoproteomics approach using functionalized covalent inhibitors, to detect active serine hydrolases across 11 E. faecium and Enterococcus lactis strains. Serine hydrolases are a big and diverse enzyme family, that includes known drug targets such as penicillin-binding proteins (PBPs), whereas other subfamilies are underexplored. Comparative gel-based ABPP using Bocillin-FL revealed strain- and growth condition dependent variations in PBP activities. Profiling with the broadly serine hydrolase-reactive fluorescent probe fluorophosphonate-TMR showed a high similarity across E. faecium clade A1 strains, but higher variation across A2 and E. lactis strains. To identify these serine hydrolases, we used a biotinylated probe analog allowing for enrichment and identification via liquid chromatography-mass spectrometry. We identified 11 largely uncharacterized targets (α,β-hydrolases, SGNH-hydrolases, phospholipases, amidases, peptidases) that are druggable and accessible in live vancomycin-resistant E. faecium E745 and may possess vital functions that are to be characterized in future studies.
粪肠球菌是一种肠道共生细菌,作为一种机会性的医院内病原体,其重要性与日俱增。它的高水平内在和获得性抗菌药耐药性正导致治疗选择的缺乏,尤其是对万古霉素耐药菌株的感染,因此需要优先鉴定和功能验证新的药物靶点。在这里,我们利用基于活性的蛋白质分析(ABPP)--一种使用功能化共价抑制剂的化学蛋白质组学方法--检测了 11 株粪肠球菌和乳酸肠球菌的活性丝氨酸水解酶。丝氨酸水解酶是一个庞大而多样化的酶家族,其中包括青霉素结合蛋白(PBPs)等已知的药物靶标,而其他亚家族则尚未得到充分探索。使用 Bocillin-FL 进行基于凝胶的 ABPP 比较显示了 PBP 活性随菌株和生长条件的变化。使用广泛的丝氨酸水解酶反应荧光探针氟膦酸盐-TMR进行的分析表明,粪肠球菌 A1 支系菌株之间的相似性很高,但 A2 和乳杆菌菌株之间的差异较大。为了鉴定这些丝氨酸水解酶,我们使用了一种生物素化探针类似物,以便通过液相色谱-质谱法进行富集和鉴定。我们在耐万古霉素的粪肠球菌 E745 中鉴定出了 11 个基本未定性的靶标(α,β-水解酶、SGNH-水解酶、磷脂酶、酰胺酶、肽酶),这些靶标可用于药物并可在活的耐万古霉素粪肠球菌 E745 中获得,而且可能具有重要功能,有待今后的研究加以鉴定。
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引用次数: 0
Colonization of vancomycin-resistant Enterococcus faecium in human-derived colonic epithelium: Unraveling the transcriptional dynamics of host-enterococcal interactions 耐万古霉素肠球菌在人源结肠上皮细胞中的定植:揭示宿主与肠球菌相互作用的转录动态
Pub Date : 2024-05-07 DOI: 10.1093/femsmc/xtae014
P. Stege, J. Beekman, Antoni P. A. Hendrickx, Laura van Eijk, Malbert R. C. Rogers, S. Suen, A. Vonk, Rob J. L. Willems, F. Paganelli
Enterococcus faecium is an opportunistic pathogen able to colonize the intestines of hospitalized patients. This initial colonization is an important step in the downstream pathogenesis, which includes outgrowth of the intestinal microbiota and potential infection of the host. The impact of intestinal overgrowth on host-enterococcal interactions is not well understood. We therefore applied a RNAseq approach in order to unravel the transcriptional dynamics of E. faecium upon co-culturing with human derived colonic epithelium. Co-cultures of colonic epithelium with a hospital-associated vancomycin resistant (vanA-type) E. faecium (VRE) showed that VRE resided on top of the colonic epithelium when analyzed by microscopy. RNAseq revealed that exposure to the colonic epithelium resulted in upregulation of 238 VRE genes compared to the control condition, including genes implicated in pili expression, conjugation (plasmid_2), genes related to sugar uptake and biofilm formation (chromossome). In total, 260 were downregulated including the vanA operon located on plasmid_3. Pathway analysis revealed an overall switch in metabolism to amino acid scavenging and reduction. In summary, our study demonstrates that co-culturing of VRE with human colonic epithelium promotes an elaborate gene response in VRE, enhancing our insight in host-E. faecium interactions, which might facilitate the design of novel anti-infectivity strategies.
粪肠球菌是一种机会性病原体,能够在住院患者的肠道中定植。最初的定植是下游发病机制的重要一步,包括肠道微生物群的生长和宿主的潜在感染。肠道过度生长对宿主与肠球菌相互作用的影响尚不十分清楚。因此,我们采用 RNAseq 方法来揭示粪肠球菌与人类结肠上皮共培养时的转录动态。将结肠上皮与医院相关的耐万古霉素(vanA 型)粪大肠杆菌(VRE)共培养后,显微镜分析显示 VRE 位于结肠上皮的顶部。RNAseq 结果显示,与对照组相比,暴露于结肠上皮导致 238 个 VRE 基因上调,其中包括与纤毛表达、连接(质粒_2)、糖摄取和生物膜形成(染色体组)相关的基因。总共有 260 个基因被下调,其中包括位于质粒_3 上的 vanA 操作子。通路分析表明,新陈代谢总体上转向氨基酸清除和还原。总之,我们的研究表明,将 VRE 与人类结肠上皮细胞共培养可促进 VRE 中复杂的基因反应,从而提高我们对宿主与粪肠杆菌相互作用的洞察力,这可能有助于设计新型抗感染策略。
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引用次数: 0
Virulence Characteristics of Gram-positive Bacteria Isolated from Diabetic Foot Ulcers 从糖尿病足溃疡中分离出的革兰氏阳性细菌的毒性特征
Pub Date : 2024-05-06 DOI: 10.1093/femsmc/xtae013
Rebecca A. Keogh, Savannah Huyvaert, Garrett D Moore, Alexander R. Horswill, K. Doran
Diabetic wound infections including diabetic foot ulcers (DFUs) are a major global health concern and a leading cause of non-traumatic amputations. Numerous bacterial species establish infection in DFUs, and treatment with antibiotics often fails due to widespread antibiotic resistance and biofilm formation. Determination of bacterial species that reside in DFU and their virulence potential is critical to inform treatment options. Here, we isolate bacteria from debridement tissues from patients with diabetes at the University of Colorado Anschutz Medical Center. The most frequent species were Gram-positive including Enterococcus faecalis, Staphylococcus aureus, and Streptococcus agalactiae, also known as Group B Streptococcus (GBS). Most tissues had more than one species isolated with E. faecalis and GBS frequently occurring in polymicrobial infection with S. aureus. S. aureus was the best biofilm producing species with E. faecalis and GBS isolates exhibiting little to no biofilm formation. Antibiotic susceptibility varied amongst strains with high levels of penicillin resistance amongst S. aureus, clindamycin resistance amongst GBS and intermediate vancomycin resistance amongst E. faecalis. Finally, we utilized a murine model of diabetic wound infection and found that the presence of S. aureus led to significantly higher recovery of GBS and E. faecalis compared to mice challenged in mono-infection.
包括糖尿病足溃疡(DFUs)在内的糖尿病伤口感染是全球关注的主要健康问题,也是非创伤性截肢的主要原因。糖尿病足溃疡中的感染细菌种类繁多,由于广泛的抗生素耐药性和生物膜的形成,抗生素治疗往往失败。确定DFU中的细菌种类及其毒力潜力对于提供治疗方案至关重要。在这里,我们从科罗拉多大学安舒茨医学中心糖尿病患者的清创组织中分离出细菌。最常见的细菌是革兰氏阳性菌,包括粪肠球菌、金黄色葡萄球菌和无乳链球菌(又称 B 群链球菌 (GBS))。大多数组织都分离出一种以上的菌种,其中粪肠球菌和 GBS 经常与金黄色葡萄球菌一起出现在多微生物感染中。金黄色葡萄球菌是最容易形成生物膜的菌种,而粪肠球菌和 GBS 分离物几乎不形成生物膜。不同菌株对抗生素的敏感性各不相同,金黄色葡萄球菌对青霉素的耐药性较高,GBS 对克林霉素的耐药性较高,而粪肠球菌对万古霉素的耐药性处于中等水平。最后,我们利用小鼠糖尿病伤口感染模型发现,与单一感染的小鼠相比,金黄色葡萄球菌的存在导致 GBS 和粪肠球菌的恢复率明显更高。
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引用次数: 0
Fungal endophytes from saline-adapted shrubs induce salinity stress tolerance in tomato seedlings. 来自适应盐碱环境的灌木的真菌内生菌诱导番茄幼苗耐盐碱胁迫。
Pub Date : 2024-04-29 eCollection Date: 2024-01-01 DOI: 10.1093/femsmc/xtae012
Priscillar Mumo Mutungi, Vitalis Wafula Wekesa, Justus Onguso, Erustus Kanga, Steve B S Baleba, Hamadi Iddi Boga

To meet the food and feed demands of the growing population, global food production needs to double by 2050. Climate change-induced challenges to food crops, especially soil salinization, remain a major threat to food production. We hypothesize that endophytic fungi isolated from salt-adapted host plants can confer salinity stress tolerance to salt-sensitive crops. Therefore, we isolated fungal endophytes from shrubs along the shores of saline alkaline Lake Magadi and evaluated their ability to induce salinity stress tolerance in tomato seeds and seedlings. Of 60 endophytic fungal isolates, 95% and 5% were from Ascomycetes and Basidiomycetes phyla, respectively. The highest number of isolates (48.3%) were from the roots. Amylase, protease and cellulase were produced by 25, 30 and 27 isolates, respectively; and 32 isolates solubilized phosphate. Only eight isolates grew at 1.5 M NaCl. Four fungal endophytes (Cephalotrichum cylindricum, Fusarium equiseti, Fusarium falciforme and Aspergilus puniceus) were tested under greenhouse conditions for their ability to induce salinity tolerance in tomato seedlings. All four endophytes successfully colonized tomato seedlings and grew in 1.5 M NaCl. The germination of endophyte-inoculated seeds was enhanced by 23%, whereas seedlings showed increased chlorophyll and biomass content and decreased hydrogen peroxide content under salinity stress, compared with controls. The results suggest that the the four isolates can potentially be used to mitigate salinity stress in tomato plants in salt-affected soils.

为了满足不断增长的人口对粮食和饲料的需求,全球粮食产量到 2050 年需要翻一番。气候变化给粮食作物带来的挑战,尤其是土壤盐碱化,仍然是粮食生产的主要威胁。我们假设,从适应盐分的寄主植物中分离出的内生真菌可以赋予对盐分敏感的作物耐盐胁迫的能力。因此,我们从盐碱地马加迪湖沿岸的灌木中分离出了内生真菌,并评估了它们诱导番茄种子和幼苗耐盐胁迫的能力。在 60 个内生真菌分离物中,分别有 95% 和 5% 来自子囊菌门和担子菌门。来自根部的分离物数量最多(48.3%)。分别有 25、30 和 27 个分离菌株产生淀粉酶、蛋白酶和纤维素酶;32 个分离菌株溶解磷酸盐。只有 8 个分离菌株在 1.5 M NaCl 条件下生长。在温室条件下测试了四种真菌内生菌(Cephalotrichum cylindricum、Fusarium equiseti、Fusarium falciforme 和 Aspergilus puniceus)诱导番茄幼苗耐盐的能力。所有四种内生菌都成功定殖了番茄幼苗,并在 1.5 M NaCl 中生长。与对照组相比,内生菌接种种子的萌发率提高了 23%,幼苗在盐胁迫下叶绿素和生物量含量增加,过氧化氢含量降低。结果表明,这四种分离物有可能用于减轻盐渍土壤中番茄植株的盐渍胁迫。
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引用次数: 0
The gut microbiome of farmed Arctic char (Salvelinus alpinus) is shaped by feeding stage and nutrient presence. 养殖北极红点鲑(Salvelinus alpinus)的肠道微生物群受饲养阶段和营养物质的影响。
Pub Date : 2024-04-23 eCollection Date: 2024-01-01 DOI: 10.1093/femsmc/xtae011
Stephen Knobloch, Sigurlaug Skirnisdóttir, Marianne Dubois, Lucie Mayolle, Laetitia Kolypczuk, Françoise Leroi, Alexandra Leeper, Delphine Passerini, Viggó Þ Marteinsson

The gut microbiome plays an important role in maintaining health and productivity of farmed fish. However, the functional role of most gut microorganisms remains unknown. Identifying the stable members of the gut microbiota and understanding their functional roles could aid in the selection of positive traits or act as a proxy for fish health in aquaculture. Here, we analyse the gut microbial community of farmed juvenile Arctic char (Salvelinus alpinus) and reconstruct the metabolic potential of its main symbionts. The gut microbiota of Arctic char undergoes a succession in community composition during the first weeks post-hatch, with a decrease in Shannon diversity and the establishment of three dominant bacterial taxa. The genome of the most abundant bacterium, a Mycoplasma sp., shows adaptation to rapid growth in the nutrient-rich gut environment. The second most abundant taxon, a Brevinema sp., has versatile metabolic potential, including genes involved in host mucin degradation and utilization. However, during periods of absent gut content, a Ruminococcaceae bacterium becomes dominant, possibly outgrowing all other bacteria through the production of secondary metabolites involved in quorum sensing and cross-inhibition while benefiting the host through short-chain fatty acid production. Whereas Mycoplasma is often present as a symbiont in farmed salmonids, we show that the Ruminococcaceae species is also detected in wild Arctic char, suggesting a close evolutionary relationship between the host and this symbiotic bacterium.

肠道微生物群在维持养殖鱼类的健康和产量方面发挥着重要作用。然而,大多数肠道微生物的功能作用仍然未知。确定肠道微生物群的稳定成员并了解它们的功能作用有助于选择积极的性状或作为水产养殖中鱼类健康的替代物。在这里,我们分析了养殖的北极红点鲑(Salvelinus alpinus)幼鱼的肠道微生物群落,并重建了其主要共生体的代谢潜力。北极红点鲑的肠道微生物群落在孵化后的最初几周内经历了群落组成的演替,香农多样性下降,并形成了三个优势细菌类群。数量最多的细菌是一种支原体,其基因组显示出对营养丰富的肠道环境中快速生长的适应。数量第二多的类群是一种 Brevinema sp.,具有多种代谢潜力,包括参与宿主粘蛋白降解和利用的基因。不过,在肠道内容物缺乏的时期,反刍球菌属细菌会成为优势菌,可能通过产生参与法定量感应和交叉抑制的次级代谢产物而超过所有其他细菌,同时通过产生短链脂肪酸而使宿主受益。支原体通常作为共生体存在于养殖的鲑鱼中,而我们的研究表明,在野生北极红点鲑中也能检测到反刍球菌,这表明宿主与这种共生细菌之间有着密切的进化关系。
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引用次数: 0
Interactions between commensal Enterococcus faecium and Enterococcus lactis and clinical isolates of E. faecium 共生粪肠球菌和乳肠球菌与临床分离的粪肠球菌之间的相互作用
Pub Date : 2024-03-09 DOI: 10.1093/femsmc/xtae009
T. Wagner, A. K. Pöntinen, Carolin Kornelia Fenzel, Daniel Engi, J. Janice, Ana C. Almeida-Santos, A. Tedim, Ana R. Freitas, Luísa Peixe, W. van Schaik, Mona Johannessen, K. Hegstad
Enterococcus faecium (Efm) is a versatile pathogen, responsible for multidrug-resistant infections, especially in hospitalized immunocompromised patients. Its population structure has been characterized by diverse clades (A1, A2 and B (reclassified as Enterococcus lactis (Ela)), adapted to different environments, and distinguished by their resistomes and virulomes. These features only partially explain the predominance of clade A1 strains in nosocomial infections. We investigated in vitro interaction of 50 clinical isolates (clade A1 Efm) against 75 commensal faecal isolates from healthy humans (25 clade A2 Efm and 50 Ela). Only 36% of the commensal isolates inhibited clinical isolates, while 76% of the clinical isolates inhibited commensal isolates. The most apparent overall differences in inhibition patterns were presented between clades. The inhibitory activity was mainly mediated by secreted, proteinaceous, heat-stable compounds, likely indicating an involvement of bacteriocins. A custom-made database targeting 76 Bacillota bacteriocins was used to reveal bacteriocins in the genomes. Our systematic screening of the interactions between nosocomial and commensal Efm and Ela on a large scale suggests that, in a clinical setting, nosocomial strains not only have an advantage over commensal strains due to their possession of AMR genes, virulence factors, and resilience but also inhibit the growth of commensal strains.
粪肠球菌(Enterococcus faecium,Efm)是一种多用途病原体,是耐多药感染的罪魁祸首,尤其是在住院的免疫力低下患者中。它的种群结构以不同的支系(A1、A2 和 B(重新分类为乳酸肠球菌(Ela))为特征,这些支系适应不同的环境,并以抗药性和毒力组来区分。这些特征只能部分解释 A1 支系菌株在医院感染中占主导地位的原因。我们研究了 50 株临床分离株(A1 族 Efm)与 75 株健康人粪便共生分离株(25 株 A2 族 Efm 和 50 株 Ela)的体外相互作用。只有 36% 的共生分离物对临床分离物有抑制作用,而 76% 的临床分离物对共生分离物有抑制作用。不同支系之间抑制模式的总体差异最为明显。抑制活性主要由分泌型、蛋白型、热稳定型化合物介导,这可能表明细菌素的参与。我们利用一个针对 76 种芽孢杆菌细菌素的定制数据库来揭示基因组中的细菌素。我们对鼻腔菌株与普通菌株 Efm 和 Ela 之间的相互作用进行了大规模的系统筛选,结果表明,在临床环境中,鼻腔菌株不仅因拥有 AMR 基因、毒力因子和恢复能力而比普通菌株更具优势,而且还能抑制普通菌株的生长。
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引用次数: 0
Mining the Microbiome of Lake Afdera to Gain Insights into Microbial Diversity and Biosynthetic Potential 挖掘阿夫德拉湖微生物组,深入了解微生物多样性和生物合成潜力
Pub Date : 2024-03-07 DOI: 10.1093/femsmc/xtae008
E. Balcha, M. Macey, M. T. Gemeda, Barbara Cavalazzi, A. A. Woldesemayat
Microorganisms inhabiting hypersaline environments have received significant attention due to their ability to thrive under poly-extreme conditions, including high salinity, elevated temperatures and heavy metal stress. They are believed to possess biosynthetic gene clusters (BGCs) that encode secondary metabolites as survival strategy and offer potential biotechnological applications. In this study, we mined BGCs in shotgun metagenomic sequences generated from Lake Afdera, a hypersaline lake in the Afar Depression, Ethiopia. The microbiome of Lake Afdera is predominantly bacterial, with Acinetobacter (18.6%) and Pseudomonas (11.8%) being ubiquitously detected. A total of 94 distinct BGCs were identified in the metagenomic data. These BGCs are found to encode secondary metabolites with two main categories of functions: (i) potential pharmaceutical applications (nonribosomal peptide synthase NRPs, polyketide synthase, others) and (ii) miscellaneous roles conferring adaptation to extreme environment (bacteriocins, ectoine, others). Notably, NRPs (20.6%) and bacteriocins (10.6%) were the most abundant. Furthermore, our metagenomic analysis predicted gene clusters that enable microbes to defend against a wide range of toxic metals, oxidative stress and osmotic stress. These findings suggest that Lake Afdera is a rich biological reservoir, with the predicted BGCs playing critical role in the survival and adaptation of extremophiles.
栖息在高盐环境中的微生物由于能够在高盐度、高温和重金属胁迫等多重极端条件下茁壮成长而备受关注。它们被认为拥有生物合成基因簇(BGCs),这些基因簇编码次生代谢物作为生存策略,并提供潜在的生物技术应用。在这项研究中,我们从埃塞俄比亚阿法尔凹地的一个高盐度湖泊阿夫德拉湖(Lake Afdera)的霰弹枪元基因组序列中挖掘出了生物合成基因簇。阿夫德拉湖的微生物组以细菌为主,其中普遍检测到醋杆菌(18.6%)和假单胞菌(11.8%)。元基因组数据共鉴定出 94 个不同的 BGCs。这些 BGCs 编码的次级代谢产物具有两大类功能:(i) 潜在的药物应用(非核糖体肽合成酶 NRPs、多酮合成酶等)和 (ii) 适应极端环境的其他作用(细菌素、外肽等)。值得注意的是,NRPs(20.6%)和细菌素(10.6%)的含量最高。此外,我们的元基因组分析还预测了使微生物能够抵御多种有毒金属、氧化应激和渗透应激的基因簇。这些研究结果表明,阿夫德拉湖是一个丰富的生物宝库,其中预测的生物基因组在嗜极端生物的生存和适应过程中发挥着关键作用。
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引用次数: 0
The gut metagenome harbors metabolic and antibiotic resistance signatures of moderate-to-severe asthma 肠道元基因组蕴藏着中重度哮喘的代谢和抗生素耐药性特征
Pub Date : 2024-03-07 DOI: 10.1093/femsmc/xtae010
Naomi G. Wilson, Ariel Hernandez-Leyva, Drew J Schwartz, Leonard B. Bacharier, A. Kau
Asthma is a common allergic airway disease that has been associated with the development of the human microbiome early in life. Both the composition and function of the infant gut microbiota have been linked to asthma risk, but functional alterations in the gut microbiota of older patients with established asthma remain an important knowledge gap. Here, we performed whole metagenomic shotgun sequencing of 95 stool samples from a cross-sectional cohort of 59 healthy and 36 subjects with moderate-to-severe asthma to characterize the metagenomes of gut microbiota in adults and children 6 years and older. Mapping of functional orthologs revealed that asthma contributes to 2.9% of the variation in metagenomic content even when accounting for other important clinical demographics. Differential abundance analysis showed an enrichment of long-chain fatty acid (LCFA) metabolism pathways which have been previously implicated in airway smooth muscle and immune responses in asthma. We also observed increased richness of antibiotic resistance genes (ARGs) in people with asthma. Several differentially abundant ARGs in the asthma cohort encode resistance to macrolide antibiotics, which are often prescribed to patients with asthma. Lastly, we found that ARG and virulence factor (VF) richness in the microbiome were correlated in both cohorts. ARG and VF pairs co-occurred in both cohorts suggesting that virulence and antibiotic resistance traits are co-selected and maintained in the fecal microbiota of people with asthma. Overall, our results show functional alterations via LCFA biosynthetic genes and increases in antibiotic resistance genes in the gut microbiota of subjects with moderate-to-severe asthma and could have implications for asthma management and treatment.
哮喘是一种常见的过敏性气道疾病,与生命早期人类微生物群的发育有关。婴儿肠道微生物群的组成和功能都与哮喘风险有关,但老年哮喘患者肠道微生物群的功能改变仍然是一个重要的知识空白。在这里,我们对来自 59 名健康人和 36 名中重度哮喘患者的 95 份横断面队列粪便样本进行了整个元基因组枪式测序,以描述成人和 6 岁及以上儿童肠道微生物群元基因组的特征。功能直向同源物图谱显示,即使考虑到其他重要的临床人口统计学因素,哮喘也占元基因组内容变化的 2.9%。差异丰度分析表明,长链脂肪酸(LCFA)代谢通路的含量丰富,这与哮喘患者的气道平滑肌和免疫反应有关。我们还观察到哮喘患者的抗生素耐药基因(ARGs)丰富度有所增加。哮喘患者队列中几个不同程度丰富的 ARGs 编码对大环内酯类抗生素的耐药性,而哮喘患者通常服用大环内酯类抗生素。最后,我们发现在两个队列中,微生物组中 ARG 和毒力因子(VF)的丰富程度是相关的。ARG和VF对在两个队列中同时出现,这表明哮喘患者粪便微生物群中的毒力和抗生素耐药性特征是共同选择和维持的。总之,我们的研究结果表明,在中重度哮喘患者的肠道微生物群中,LCFA 生物合成基因发生了功能性改变,抗生素耐药性基因增加,这可能对哮喘的管理和治疗产生影响。
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引用次数: 0
Comparative assessment of Nanotrap and polyethylene glycol-based virus concentration in wastewater samples 废水样本中纳米诱捕器和聚乙二醇病毒浓度的比较评估
Pub Date : 2024-03-05 DOI: 10.1093/femsmc/xtae007
K. Farkas, J. Kevill, Rachel C Williams, Igor Pântea, Nicola Ridding, Kathryn Lambert-Slosarska, Nick Woodhall, Jasmine M S Grimsley, Matthew J. Wade, Andrew C. Singer, Andrew J Weightman, Gareth Cross, Davey L Jones
Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4–21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing.
以废水为基础的流行病学目前已在许多国家广泛应用,用于在社区一级对 SARS-CoV-2 和其他病毒进行常规监测。然而,高效的样本处理技术仍在研究之中。在本研究中,我们比较了新型 Nanotrap® 微生物组微粒(NMP)浓缩法与常用的聚乙二醇(PEG)沉淀法在浓缩废水中的病毒及其后续定量和测序方面的性能。为此,我们首先在废水中添加了 SARS-CoV-2、流感病毒、麻疹病毒和诺如病毒,结果发现,根据病毒类型的不同,NMP 方法可回收 0.4-21%的病毒,且结果一致、可重复。利用 NMP 和 PEG 方法,我们使用基于定量 PCR (qPCR) 的方法和新一代测序技术监测了废水中的 SARS-CoV-2、甲型和乙型流感病毒、RSV、肠道病毒、诺如病毒 GI 和 GII 以及 crAssphage。这两种方法都能很好地回收高丰度病毒,但 PEG 沉淀法在回收废水中低丰度病毒方面更为成功。此外,用 PEG 沉淀法处理的样本比用 NMP 法处理的样本更能成功地对 SARS-CoV-2 进行测序。使用 PEG 沉淀法时,样品量越大,病毒回收率越高。总之,我们的研究结果表明,NMP 浓缩法是一种快速简便的病毒浓缩方法,适用于废水中含量丰富的病毒目标物,而 PEG 沉淀法可能更适用于低含量病毒的回收和分析以及下一代测序。
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引用次数: 0
Prevalence of diverse antimicrobial resistance genes and bacteria in sewage treatment plant-derived sludge environment. 污水处理厂污泥环境中各种抗菌药耐药性基因和细菌的流行情况。
Pub Date : 2024-02-28 eCollection Date: 2024-01-01 DOI: 10.1093/femsmc/xtae004
Mrinmoy Patra, Bhavana Pandey, Suresh Kumar Dubey

Antimicrobial resistance (AMR) contamination in the environment is one of the most significant worldwide threats of the 21st century. Since sludge is heavily exposed to diverse contaminants, including pharmaceuticals, the inhabitant bacterial population is expected to exhibit resistance to antimicrobial agents. In this study, sewage treatment plant (STP) sludge samples were analyzed to assess the antibiotic-resistant bacterial population, abundance of AMR genes (ermF, qnrS, Sul1, blaGES, blaCTX-M, and blaNDM), and mobile genetic elements (intl1 and IS26). Out of 16, six bacterial isolates exhibited resistance to 13 antibiotics with a high multiple antibiotic resistance index (MARI) (0.93) and high metal tolerance. Quantitative polymerase chain reaction showed the abundance of target genes ranging from 6.6 × 103 to 6.5 × 108 copies g-1 sludge. The overall outcome reveals that STP sludge comprised varied multidrug-resistant bacterial populations. It will give insights into the functions of heavy metals and biofilm development in the selection and spread of AMR genes and the associated bacteria. Therefore, the application of sludge needs proper screening for AMR and metal contamination prior to its countless applications. This study will contribute immensely to the risk analysis of STP effluents on environmental health, including control of AMR transmission.

环境中的抗菌剂耐药性(AMR)污染是 21 世纪全球最重大的威胁之一。由于污泥大量暴露于包括药物在内的各种污染物中,因此预计污泥中的细菌种群会表现出对抗菌剂的耐药性。本研究对污水处理厂(STP)污泥样本进行了分析,以评估抗生素耐药细菌的数量、AMR 基因(ermF、qnrS、Sul1、blaGES、blaCTX-M 和 blaNDM)以及移动遗传因子(intl1 和 IS26)的丰度。在 16 个细菌分离物中,有 6 个细菌对 13 种抗生素具有耐药性,多重抗生素耐药性指数(MARI)高达 0.93,并具有较强的金属耐受性。定量聚合酶链反应显示,目标基因的丰度范围为 6.6 × 103 至 6.5 × 108 拷贝 g-1 污泥。总体结果表明,污泥处理厂污泥中含有多种耐多药细菌种群。这将有助于深入了解重金属和生物膜发展在选择和传播 AMR 基因及相关细菌方面的功能。因此,污泥的应用需要在无数次应用之前进行适当的 AMR 和金属污染筛查。这项研究将为污水处理厂污水对环境健康的风险分析做出巨大贡献,包括控制 AMR 的传播。
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FEMS microbes
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