bluuebunny/crossref_metadata_2025 huggingface dataset:
A Saga-In-Progress: Challenges and Milestones on Our Way Toward the Nordic Core Values and Principles of Family Medicine/General Practice
Authors: Sigurdsson Johann A. , Beich Anders , Stavdal Anna
Published: December 2021

Summary: Late in 2020, the Nordic Colleges of General Practice published a joint statement specifying what General Practitioners stand for and intend to act upon, our Core Values and Principles. In this article, the authors describe and analyze challenges and milestones encountered on our 50-year journey toward the creation of that document.

The shaping of Family Medicine/General Practice as an academic discipline began in the 1960's. During an initial, descriptive phase, the new specialty was defined, its educational curricula formulated, and the core competencies required to earn the title, Specialist in Family Medicine, were identified. Focus was not yet placed directly on the relationship between viable working principles and values, however.

Then, the 1978 WHO Alma Ata Declaration affirmed health to be a fundamental human right, with primary health care as the heart of sustainable health care systems, indirectly mandating that the field of Family Medicine deliver value-based health care. A major step in that process was taken in 2001: The Norwegian College of General Practice launched their statement identifying the seven theses, Sju teser, that characterize the principles, purposes—and core values—of General Practice. Later, the Nordic colleges worked together to formulate the 2020 joint statement.

We are confident that Family Medicine will continue to provide sustainable, relationship-based care, and to protect the human side of medicine. Sharing core values and principles can help us mobilize as effective advocates for our discipline and for our patients, the citizens whom we serve.
Case Report: Tocilizumab for Acute Kidney Graft Dysfunction in Patient Affected by COVID-19
Authors: Barbara Infante , Silvia Mercuri , Dario Troise , Giuseppe Castellano , Stallone Giovanni
Published: December 2021

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was isolated in January 2020 and, on March, the WHO declared the status of a pandemic. It causes a cytokine release syndrome, called “cytokine storm,” characterized by systemic inflammation involving elevated levels of cytokines and hyperactivation of immune cell; this profound alteration in the immune system led to an overshooting inflammatory response contributing to morbidity and mortality. Solid organ transplant recipients are at particularly higher risk of developing critical coronavirus disease 2019 (COVID-19) due to chronic immunosuppression; in fact, establishing the balance between infection and rejection in any transplant recipient is the principal aim when prescribing immunosuppression. Tocilizumab, a humanized monoclonal antibody against interleukin-6 (IL-6) receptor widely adopted in adult rheumatoid arthritis, is used as rescue therapy for chronic antibody-mediated rejection in kidney transplantation. Data about the use of tocilizumab for treating acute kidney graft dysfunction in a setting of kidney-transplanted patients affected by COVID-19 are lacking. In this case study, we discuss the case of kidney transplant recipient with proven SARS-CoV-2 infection that develops acute graft dysfunction and the management of immunosuppression with concomitant tocilizumab administration.
Choroidal Neovascularization in Pediatric Patients: Analysis of Etiologic Factors, Clinical Characteristics and Treatment Outcomes
Authors: Zhang Ting , Wang You , Yan Wenjia , Liu Yafen , Lu Jinglin , Sun Limei , Li Songshan , Huang Li , Zhang Zhaotian , Ding Xiaoyan
Published: December 2021

Background and Objectives: Choroidal neovascularization (CNV) is a common pathologic lesion that occurs in various chorioretinopathy, but very limited published data have reported in pediatric patients. This study aimed to investigate the etiologic factors, clinical features, and treatment outcomes of choroidal neovascularization (CNV) in children.

Methods: In this study, 33 eyes in 30 patients aged 18 years or younger with CNV were included. Comprehensive ophthalmic examination was performed in all the patients. The demographic profiles, laterality, visual acuity, optical coherence tomographic findings, fundus fluorescein angiographic findings, and the underlying pathology were analyzed. The types, locations, treatment outcomes, and recurrences of CNV were noted.

Results: The average age was 11.2 ± 4.6 (range, 1–18) years. Most CNVs affecting children were classic and type 2. The most common etiologic factors of CNV in pediatric patients were congenital/developing abnormalities (9/30, 30.0%) and inflammatory retinochoroidopathy (9/30, 30.0%), followed by idiopathic CNV (8/30, 26.7%). Subtype analysis showed that the etiologic factor was inflammatory retinochoroidopathy in children 12 years or older, whereas congenital/developing abnormalities were present in children younger than 12 years. Eyes with active CNVs required a mean of 1.40 ± 0.58 injections. No recurrence was observed during follow-up.

Conclusions: The etiologic factors of CNV in young Chinese patients were diverse, with congenital/developing abnormalities, inflammatory retinochoroidopathy and idiopathic CNV being the 3 most common ones. Eyes with active CNVs had good responses to antivascular endothelial growth factor treatment with low recurrence.
Chronic Hematuria Increases Chronic Kidney Injury and Epithelial–Mesenchymal Transition in 5/6 Nephrectomy Rats
Authors: Xiao Min , Medipally Ajay K. , Biederman Laura , Satoskar Anjali A. , Ivanov Iouri , Rovin Brad H. , Brodsky Sergey V.
Published: December 2021

Chronic kidney disease (CKD) is a common outcome of many kidney diseases. Interstitial fibrosis and tubular atrophy (IFTA) is a histologic hallmark of CKD. Hematuria is a common symptom in many human kidney diseases. Free hemoglobin may affect tubular epithelial cells by generating reactive oxygen species (ROS). Epithelial–mesenchymal transition (EMT) of the tubular epithelial cells has been shown to play an important role in the IFTA development. The aim of this study was to determine the effects of chronic hematuria on the CKD progression in 5/6 nephrectomy (5/6NE) rat model of CKD. 5/6 NE rats were treated with oral warfarin (0.5 mg/kg/day) or vehicle (control). The animals were monitored for 26 weeks, while prothrombin time (PT), serum creatinine (SCr), and hematuria were measured weekly. Staining for iron, trichrome, and EMT (vimentin, E-cadherin, smooth muscle actin) markers was performed on the remnant kidneys. ROS were detected in the kidneys by protein carbonyl assay and immunohistochemistry for heme oxygenase 1 (HMOX1), at the end of the study. Apoptosis was detected by TUNEL assay. Warfarin treatment resulted in a PT increase 1.5–2.5 times from control and an increase in hematuria and SCr. Histologically, warfarin-treated animals had more iron-positive tubular epithelial cells and increased IFTA as compared to control (42.9 ± 17% vs. 18.3 ± 2.6%). ROS were increased in the kidney in warfarin-treated rats. The number of tubules that show evidence of EMT was significantly higher in warfarin-treated 5/6NE as compared to control 5/6NE rats. The number of apoptotic tubular epithelial cells was higher in warfarin-treated 5/6 NE rats. Chronic hematuria results in increased iron-positive tubular epithelial cells, EMT, apoptosis, and more prominent IFTA in CKD rats. Our data suggest an important role of chronic hematuria in the progression of CKD.
Intraoperative Fractions of Inspiratory Oxygen Are Associated With Recurrence-Free Survival After Elective Cancer Surgery
Authors: Dehne Sarah , Spang Verena , Klotz Rosa , Kummer Laura , Kilian Samuel , Hoffmann Katrin , Schneider Martin A. , Hackert Thilo , Büchler Markus W. , Weigand Markus A. , Larmann Jan
Published: December 2021

Background: Choice of the fraction of inspiratory oxygen (FiO2) is controversial. The objective of this analysis was to evaluate whether intraoperative FiO2 was associated with recurrence-free survival after elective cancer surgery.

Methods and Analysis: In this single-center, retrospective study, we analyzed 1,084 patients undergoing elective resection of pancreatic (n = 652), colorectal (n = 405), or hepatic cancer (n = 27) at Heidelberg University Hospital between 2009 and 2016. Intraoperative mean FiO2 values were calculated. For unstratified analyses, the study cohort was equally divided into a low- and a high-FiO2 group. For cancer-stratified analyses, this division was done within cancer-strata. The primary outcome measure was recurrence-free survival until the last known follow-up. Groups were compared using Kaplan–Meier analysis. A stratified log rank test was used to control for different FiO2 levels and survival times between the cancer strata. Cox-regression analyses were used to control for covariates. Sepsis, reoperations, surgical-site infections, and cardiovascular events during hospital stay and overall survival were secondary outcomes.

Results: Median FiO2 was 40.9% (Q1–Q3, 38.3–42.9) in the low vs. 50.4% (Q1–Q3, 47.4–54.7) in the high-FiO2 group. Median follow-up was 3.28 (Q1–Q3, 1.68–4.97) years. Recurrence-free survival was considerable higher in the high-FiO2 group (p < 0.001). This effect was also confirmed when stratified for the different tumor entities (p = 0.007). In colorectal cancer surgery, increased FiO2 was independently associated with increased recurrence-free survival. The hazard for the primary outcome decreased by 3.5% with every 1% increase in FiO2. The effect was not seen in pancreatic cancer surgery and we did not find differences in any of the secondary endpoints.

Conclusions: Until definite evidence from large-scale trials is available and in the absence of relevant clinical conditions warranting specific FiO2 values, perioperative care givers should aim for an intraoperative FiO2 of 50% in abdominal cancer surgery as this might benefit oncological outcomes.
Precision and Agreement of Individual and Simultaneous Macular and Optic Disc Volumetric Measurements With Spectral Domain Optical Coherence Tomography
Authors: Domínguez-Vicent Alberto , Kensén Jesper , Ramsay Marika Wahlberg , Brautaset Rune , Venkataraman Abinaya Priya
Published: December 2021

Purpose: To evaluate the precision of individual and combined macula and optic disc volumetric analysis, and the agreement between these two scan modes with spectral domain optical coherence tomography (OCT).

Methods: Macular and optic disc volumetric measurements were performed with individual and combined scan protocols in one eye of 75 healthy subjects. Three repeated measurements were performed with each protocol. From the macular area, retinal thickness in nine different sectors and ganglion cell complex thickness in eight different sectors were analyzed from both scan modes. From the optic disc area, the peripapillary retinal nerve fiber layer (pRNFL) thickness in 12 clock sectors and the optic disc parameters were evaluated. For all the parameters, repeatability limit and agreement analysis were performed.

Results: For the retinal thickness measurements in macula, the combined scan had two to three times larger repeatability limit than the individual scan for all the sectors except the central sector, where the repeatability limit was five times larger. The limits of agreement intervals were lower than 20 μm for all sectors, except the central. The ganglion cell complex measurements also had larger repeatability limits for the combined scans, and the limits of agreement intervals were <10 μm for all sectors. For the pRNFL thickness, the repeatability values were distributed like a vertically elongated ellipse for both scans, but still the repeatability was better for individual scan compared to the combined scan. The shortest and widest interval are obtained for sectors 9 (9 μm) and 12 (40 μm), respectively. The repeatability limit was <0.15 units for all disc parameters with both scan modes.

Conclusion: The individual macula and optic disc scans had better repeatability than the combined scan mode, and the two scan modes cannot be used interchangeability due to the wide limits of agreement.
The Glomerular Endothelium Restricts Albumin Filtration
Authors: Ballermann Barbara J. , Nyström Jenny , Haraldsson Börje
Published: December 2021

Inflammatory activation and/or dysfunction of the glomerular endothelium triggers proteinuria in many systemic and localized vascular disorders. Among them are the thrombotic microangiopathies, many forms of glomerulonephritis, and acute inflammatory episodes like sepsis and COVID-19 illness. Another example is the chronic endothelial dysfunction that develops in cardiovascular disease and in metabolic disorders like diabetes. While the glomerular endothelium is a porous sieve that filters prodigious amounts of water and small solutes, it also bars the bulk of albumin and large plasma proteins from passing into the glomerular filtrate. This endothelial barrier function is ascribed predominantly to the endothelial glycocalyx with its endothelial surface layer, that together form a relatively thick, mucinous coat composed of glycosaminoglycans, proteoglycans, glycolipids, sialomucins and other glycoproteins, as well as secreted and circulating proteins. The glycocalyx/endothelial surface layer not only covers the glomerular endothelium; it extends into the endothelial fenestrae. Some glycocalyx components span or are attached to the apical endothelial cell plasma membrane and form the formal glycocalyx. Other components, including small proteoglycans and circulating proteins like albumin and orosomucoid, form the endothelial surface layer and are bound to the glycocalyx due to weak intermolecular interactions. Indeed, bound plasma albumin is a major constituent of the endothelial surface layer and contributes to its barrier function. A role for glomerular endothelial cells in the barrier of the glomerular capillary wall to protein filtration has been demonstrated by many elegant studies. However, it can only be fully understood in the context of other components, including the glomerular basement membrane, the podocytes and reabsorption of proteins by tubule epithelial cells. Discovery of the precise mechanisms that lead to glycocalyx/endothelial surface layer disruption within glomerular capillaries will hopefully lead to pharmacological interventions that specifically target this important structure.
Case Report: Amyloidosis Cutis Dyschromica: Dermoscopy and Reflectance Confocal Microscopy and Gene Mutation Analysis of a Chinese Pedigree
Authors: Wang Hui , Zhong Zhenyu , Wang Xiuli , Zheng Liyun , Wang Yifan , Wang Shan , Liu Siqi , Li Hui , Guo Ze , Gao Min
Published: December 2021

Background: Amyloidosis cutis dyschromica (ACD) is a rare type of primary localized cutaneous amyloidosis. Non-invasive techniques can provide important clues for early diagnosis.

Objectives: To highlight the characteristic imaging changes of ACD under dermoscopy and reflectance confocal microscopy (RCM), investigate gene mutations in a Chinese Han pedigree of ACD, and analyze the genotype–phenotype correlation.

Methods: Dermoscopy and RCM examinations were completed together for the pedigree, and the imaging characteristics were described. The diagnosis of ACD was confirmed by pathological examination. Sequencing was performed followed by bioinformatics and genotype–phenotype correlation. ACD-related articles published on PubMed between January 1970 and March 2021 were reviewed and summarized.

Results: In ACD, dermoscopy showed patchy white hypopigmentation and brownish spots, stripes, or hyperpigmented blotches and patches. RCM showed a highly refractive substance with clumpy, dotted, and linear structures inside the papillary dermis. Sequencing identified glycoprotein non-metastatic melanoma protein B (GPNMB) missense mutations [c.393T>G (p.Y131X; NM_001005340.2)] and a frameshift deletion mutation [c.719_720delTG (p.V240fs; NM_001005340.2)]. The ANNOtate VARiation (ANNOVAR) software predicted that c.393T>G is a pathogenic mutation. The literature review found 14 mutations, namely, 5 (35.7%) frameshift mutations, 4 (28.6%) non-sense mutations, 4 (28.6%) missense mutations, and 1 (7.1%) splice site mutation. Blisters and epidermolysis were observed in several cases, but there was no significant association between clinical manifestations and mutations in ACD.

Conclusions: This study was the first to combine dermoscopy and RCM to describe ACD. Two GPNMB gene mutations were reported in a Chinese ACD pedigree. The genotype–phenotype correlation was analyzed for the first time; however, there was no significant correlation.
Serum CYR61 Is Associated With Airway Inflammation and Is a Potential Biomarker for Severity in Chronic Obstructive Pulmonary Disease
Authors: Tan Zhu-Xia , Fu Lin , Wang Wen-Jing , Zhan Ping , Zhao Hui , Wang Hua , Xu De-Xiang
Published: December 2021

Background: Cysteine-rich 61 (CYR61) and inflammation was upregulated in the lungs of patients with chronic obstructive pulmonary disease (COPD). However, the association between CYR61 and inflammation was unclear in COPD patients. This study aimed to analyze the association of serum CYR61 with pulmonary inflammation and lung function indexes in COPD patients.

Methods: One hundred and fifty COPD patients and 150 control subjects were enrolled. Serum and pulmonary CYR61 was detected. Lung function indexes were evaluated in COPD patients.

Results: Serum CYR61 level was elevated and pulmonary CYR61 expression was upregulated in COPD patients. An increased CYR61 was associated with decreased pulmonary function indexes in COPD patients. Further analyses showed that nuclear factor-kappa B (NF-κB) p65-positive nuclei was elevated in the lungs of COPD patients with high level of CYR61. Accordingly, serum monocyte chemotactic protein (MCP)-1 and tumor necrosis factor α (TNF-α), two downstream inflammatory cytokines of NF-κB pathway, were increased in parallel with CYR61, among which serum MCP-1 and TNF-α were the highest in COPD patients with high level of CYR61. Moreover, a positive correlation, determined by multivariate regression that excluded the influence of age, gender and smoking, was observed between serum CYR61 and inflammatory cytokines in COPD patients.

Conclusion: These results provide evidence that an increased CYR61 is associated with pulmonary inflammation and COPD progression. Inflammatory cytokines may be the mediators between CYR61 elevation and COPD progression.
Epidemiology of Extended-Spectrum Beta-Lactamase and Carbapenemase-Producing Enterobacterales in the Greater Mekong Subregion: A Systematic-Review and Meta-Analysis of Risk Factors Associated With Extended-Spectrum Beta-Lactamase and Carbapenemase Isolation
Authors: Singh Shweta R. , Teo Alvin Kuo Jing , Prem Kiesha , Ong Rick Twee-Hee , Ashley Elizabeth A. , van Doorn H. Rogier , Limmathurotsakul Direk , Turner Paul , Hsu Li Yang
Published: December 2021

Background: Despite the rapid spread of extended-spectrum beta-lactamase (ESBL) producing-Enterobacterales (ESBL-E) and carbapenemase-producing Enterobacterales (CPE), little is known about the extent of their prevalence in the Greater Mekong Subregion (GMS). In this systematic review, we aimed to determine the epidemiology of ESBL-E and CPE in clinically significant Enterobacterales: Escherichia coli and Klebsiella pneumoniae from the GMS (comprising of Cambodia, Laos, Myanmar, Thailand, Vietnam and Yunnan province and Guangxi Zhuang region of China).

Methods: Following a list of search terms adapted to subject headings, we systematically searched databases: Medline, EMBASE, Scopus and Web of Science for articles published on and before October 20th, 2020. The search string consisted of the bacterial names, methods involved in detecting drug-resistance phenotype and genotype, GMS countries, and ESBL and carbapenemase detection as the outcomes. Meta-analyses of the association between the isolation of ESBL from human clinical and non-clinical specimens were performed using the “METAN” function in STATA 14.

Results: One hundred and thirty-nine studies were included from a total of 1,513 identified studies. Despite the heterogeneity in study methods, analyzing the prevalence proportions on log-linear model scale for ESBL producing-E. coli showed a trend that increased by 13.2% (95%CI: 6.1–20.2) in clinical blood specimens, 8.1% (95%CI: 1.7–14.4) in all clinical specimens and 17.7% (95%CI: 4.9–30.4) increase in carriage specimens. Under the log-linear model assumption, no significant trend over time was found for ESBL producing K. pneumoniae and ESBL-E specimens. CPE was reported in clinical studies and carriage studies past 2010, however a trend could not be determined because of the small dataset. Twelve studies were included in the meta-analysis of risk factors associated with isolation of ESBL. Recent antibiotic exposure was the most studied variable and showed a significant positive association with ESBL-E isolation (pooled OR: 2.9, 95%CI: 2.3–3.8) followed by chronic kidney disease (pooled OR: 4.7, 95%CI: 1.8–11.9), and other co-morbidities (pooled OR: 1.6, 95%CI: 1.2–2.9).

Conclusion: Data from GMS is heterogeneous with significant data-gaps, especially in community settings from Laos, Myanmar, Cambodia and Yunnan and Guangxi provinces of China. Collaborative work standardizing the methodology of studies will aid in better monitoring, surveillance and evaluation of interventions across the GMS.
Kinetic Properties of Microbial Exoenzymes Vary With Soil Depth but Have Similar Temperature Sensitivities Through the Soil Profile
Authors: Alves Ricardo J. Eloy , Callejas Ileana A. , Marschmann Gianna L. , Mooshammer Maria , Singh Hans W. , Whitney Bizuayehu , Torn Margaret S. , Brodie Eoin L.
Published: December 2021

Current knowledge of the mechanisms driving soil organic matter (SOM) turnover and responses to warming is mainly limited to surface soils, although over 50% of global soil carbon is contained in subsoils. Deep soils have different physicochemical properties, nutrient inputs, and microbiomes, which may harbor distinct functional traits and lead to different SOM dynamics and temperature responses. We hypothesized that kinetic and thermal properties of soil exoenzymes, which mediate SOM depolymerization, vary with soil depth, reflecting microbial adaptation to distinct substrate and temperature regimes. We determined the Michaelis-Menten (MM) kinetics of three ubiquitous enzymes involved in carbon (C), nitrogen (N) and phosphorus (P) acquisition at six soil depths down to 90 cm at a temperate forest, and their temperature sensitivity based on Arrhenius/Q10 and Macromolecular Rate Theory (MMRT) models over six temperatures between 4–50°C. Maximal enzyme velocity (Vmax) decreased strongly with depth for all enzymes, both on a dry soil mass and a microbial biomass C basis, whereas their affinities increased, indicating adaptation to lower substrate availability. Surprisingly, microbial biomass-specific catalytic efficiencies also decreased with depth, except for the P-acquiring enzyme, indicating distinct nutrient demands at depth relative to microbial abundance. These results suggested that deep soil microbiomes encode enzymes with intrinsically lower turnover and/or produce less enzymes per cell, reflecting distinct life strategies. The relative kinetics between different enzymes also varied with depth, suggesting an increase in relative P demand with depth, or that phosphatases may be involved in C acquisition. Vmax and catalytic efficiency increased consistently with temperature for all enzymes, leading to overall higher SOM-decomposition potential, but enzyme temperature sensitivity was similar at all depths and between enzymes, based on both Arrhenius/Q10 and MMRT models. In a few cases, however, temperature affected differently the kinetic properties of distinct enzymes at discrete depths, suggesting that it may alter the relative depolymerization of different compounds. We show that soil exoenzyme kinetics may reflect intrinsic traits of microbiomes adapted to distinct soil depths, although their temperature sensitivity is remarkably uniform. These results improve our understanding of critical mechanisms underlying SOM dynamics and responses to changing temperatures through the soil profile.
Alterations in Arbuscular Mycorrhizal Community Along a Chronosequence of Teak (Tectona grandis) Plantations in Tropical Forests of China
Authors: Yu Zhi , Liang Kunnan , Wang Xianbang , Huang Guihua , Lin Mingping , Zhou Zaizhi , Chen Yinglong
Published: December 2021

Arbuscular mycorrhizal (AM) fungi play a crucial role in promoting plant growth, enhancing plant stress resistance, and sustaining a healthy ecosystem. However, little is known about the mycorrhizal status of teak plantations. Here, we evaluated how the AM fungal communities of rhizosphere soils and roots respond to different stand ages of teak: 22, 35, 45, and 55-year-old from the adjacent native grassland (CK). A high-throughput sequencing method was used to compare the differences in soil and root AM fungal community structures. In combination with soil parameters, mechanisms driving the AM fungal community were revealed by redundancy analysis and the Mantel test. Additionally, spore density and colonization rates were analyzed. With increasing stand age, the AM fungal colonization rates and spore density increased linearly. Catalase activity and ammonium nitrogen content also increased, and soil organic carbon, total phosphorous, acid phosphatase activity, available potassium, and available phosphorus first increased and then decreased. Stand age significantly changed the structure of the AM fungal community but had no significant impact on the diversity of the AM fungal community. However, the diversity of the AM fungal community in soils was statistically higher than that in the roots. In total, nine and seven AM fungal genera were detected in the soil and root samples, respectively. The majority of sequences in soils and roots belonged to Glomus. Age-induced changes in soil properties could largely explain the alterations in the structure of the AM fungal community along a chronosequence, which included total potassium, carbon-nitrogen ratio, ammonium nitrogen, catalase, and acid phosphatase levels in soils and catalase, acid phosphatase, pH, and total potassium levels in roots. Soil nutrient availability and enzyme activity were the main driving factors regulating the shift in the AM fungal community structure along a chronosequence of the teak plantations.
Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals
Authors: Epstein Hannah E. , Hernandez-Agreda Alejandra , Starko Samuel , Baum Julia K. , Vega Thurber Rebecca
Published: December 2021

16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given that the same variable region of the 16S gene is targeted, it is generally accepted that sequencing output from differing protocols are comparable and this assumption underlies our ability to identify universal patterns in microbial dynamics through meta-analyses. However, discrepant results from a combined 16S rRNA gene dataset prepared by two labs whose protocols differed only in DNA polymerase and sequencing platform led us to scrutinize the outputs and challenge the idea of confidently combining them for standard microbiome analysis. Using technical replicates of reef-building coral samples from two species, Montipora aequituberculata and Porites lobata, we evaluated the consistency of alpha and beta diversity metrics between data resulting from these highly similar protocols. While we found minimal variation in alpha diversity between platform, significant differences were revealed with most beta diversity metrics, dependent on host species. These inconsistencies persisted following removal of low abundance taxa and when comparing across higher taxonomic levels, suggesting that bacterial community differences associated with sequencing protocol are likely to be context dependent and difficult to correct without extensive validation work. The results of this study encourage caution in the statistical comparison and interpretation of studies that combine rRNA gene sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences.
A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits
Authors: Raguseo Celeste , Gerin Donato , Pollastro Stefania , Rotolo Caterina , Rotondo Palma Rosa , Faretra Francesco , De Miccolis Angelini Rita Milvia
Published: December 2021

Brown rot, caused by different Monilinia species, is a most economically important disease of pome and stone fruits worldwide. In Europe and in Italy, the quarantine pathogen M. fructicola was recently introduced and rapidly spread and, by competing with the main indigenous species Monilinia fructigena and Monilinia laxa, caused relevant changes in Monilinia populations. As a result, in most areas, the pathogen almost replaced M. fructigena and now coexists with M. laxa. The availability of specific and easy-of-use quantification methods is essential to study the population dynamics, and in this work, a new method for the simultaneous quantification of M. fructicola and M. laxa based on droplet digital PCR (ddPCR) technique was established. Under the optimized reaction conditions, consisting of 250/500 nM of primers/probe sets concentration, 58°C as annealing temperature and 50 PCR cycles, the duplex-ddPCR assay was 200-fold more sensitive than duplex-real-time quantitative PCR (qPCR) assay, quantifying < 1 copy μL–1 of target DNA in the PCR mixture. The results obtained with the validation assay performed on apricot and peach fruits, artificially inoculated with conidial suspensions containing different ratios of M. fructicola and M. laxa, showed a high correlation (R2 = 0.98) between the relative quantity of DNA of the two species quantified by ddPCR and qPCR and a more accurate quantification by ddPCR compared to qPCR at higher concentrations of M. fructicola. The herein described method represents a useful tool for the early detection of Monilinia spp. on stone fruits and for the improving knowledge on the epidemiology of brow rot and interactions between the two prevalent Monilinia species.
Effect of Light and p-Coumaric Acid on the Growth and Expression of Genes Related to Oxidative Stress in Brettanomyces bruxellensis LAMAP2480
Authors: Catrileo Daniela , Moreira Sandra , Ganga María Angélica , Godoy Liliana
Published: December 2021

Brettanomyces bruxellensisis considered the most significant contaminant yeast in the wine industry since it causes a deterioration in the organoleptic properties of the wine and significant economic losses. This deterioration is due to the production of volatile phenols from hydroxycinnamic acids. These compounds possess antimicrobial properties; however,B. bruxellensiscan resist this effect because it metabolizes them into less toxic ones. Recent studies have reported thatB. bruxellensisgrows under different stress conditions, includingp-coumaric acid (pCA) but effective methods for its control have not been found yet. Since that in other yeasts, such asSaccharomyces cerevisiae, it has been described that light affects its growth, and we evaluated whether the light would have a similar effect onB. bruxellensis. The results show that at light intensities of 2,500 and 4,000 lux in the absence ofpCA,B. bruxellensisLAMAP2480 does not grow in the culture medium; however, when the medium contains this acid, the yeast adapts to both factors of stress managing to grow. The expression of genes related to oxidative stress inB. bruxellensisLAMAP2480, such asSOD1,GCN4, andESBP6, showed a higher relative expression when the yeast was exposed to 2,500 lux compared to 4,000 lux, agreeing with the growth curves. This suggests that a higher expression of the genes studied would be related to stress-protective effects bypCA.
Sex Differences in Fecal Microbiota Correlation With Physiological and Biochemical Indices Associated With End-Stage Renal Disease Caused by Immunoglobulin a Nephropathy or Diabetes
Authors: Zhang Peng , Fang Jiali , Li Guanghui , Zhang Lei , Lai Xingqiang , Xu Lu , Liu Luhao , Xiong Yunyi , Li Li , Zhang Tao , Wan Jiao , Xu Hailin , Chen Rongxin , Zhang Weiting , Ma Junjie , Chen Zheng
Published: December 2021

This study investigated the sex-specific differences in the correlation between intestinal microbiota and end-stage renal disease. Here, we compared the differences in the gut microbiota of male and female healthy controls (HC) and patients with end-stage renal disease (ESRD) caused by immunoglobulin A (IgA) nephropathy (ESRD-IgAN) or type-2 diabetes mellitus (ESRD-T2DM) using high-throughput sequencing of the 16S rRNA gene. We also analyzed the correlation between gut microbiota and clinical immune indicators. We assigned 8, 10, 5, 7, 11, and 20 volunteers to female HC, ESRD-IgAN, and ESRD-T2DM, and male HC, ESRD-IgAN, and ESRD-T2DM, respectively. The results showed sex-specific differences in both physiological and biochemical indices and intestinal microbiota composition, as well as the correlation between them. The correlations between physiological and biochemical indices in men were significantly lower than those in women, especially for indices related to immunity, blood glucose, and cardiac color sonography. Urine output, lymphocyte ratio, serum albumin, blood calcium, dialysis status, serum urea nitrogen, urine protein, and diabetes significantly correlated with male fecal microbiota composition, whereas only creatinine and 2-h post-prandial blood glucose significantly correlated with female fecal microbiota composition. The top 50 dominant operational taxonomic units showed a stronger correlation with physiological and biochemical indices in samples obtained from females than from males. These differences highlight sex-specific differences in the effectiveness of ESRD prevention and treatments via regulating intestinal microbiota.
Agrobacterium tumefaciens Type IV and Type VI Secretion Systems Reside in Detergent-Resistant Membranes
Authors: Czolkoss Simon , Safronov Xenia , Rexroth Sascha , Knoke Lisa R. , Aktas Meriyem , Narberhaus Franz
Published: December 2021

Cell membranes are not homogenous but compartmentalized into lateral microdomains, which are considered as biochemical reaction centers for various physiological processes in eukaryotes and prokaryotes. Due to their special lipid and protein composition, some of these microdomains are resistant to treatment with non-ionic detergents and can be purified as detergent-resistant membranes (DRMs). Here we report the proteome of DRMs from the Gram-negative phytopathogen Agrobacterium tumefaciens. Using label-free liquid chromatography-tandem mass spectrometry, we identified proteins enriched in DRMs isolated under normal and virulence-mimicking growth conditions. Prominent microdomain marker proteins such as the SPFH (stomatin/prohibitin/flotillin/HflKC) proteins HflK, HflC and Atu3772, along with the protease FtsH were highly enriched in DRMs isolated under any given condition. Moreover, proteins involved in cell envelope biogenesis, transport and secretion, as well as motility- and chemotaxis-associated proteins were overrepresented in DRMs. Most strikingly, we found virulence-associated proteins such as the VirA/VirG two-component system, and the membrane-spanning type IV and type VI secretion systems enriched in DRMs. Fluorescence microscopy of the cellular localization of both secretion systems and of marker proteins was in agreement with the results from the proteomics approach. These findings suggest that virulence traits are micro-compartmentalized into functional microdomains in A. tumefaciens.
Co-occurrence of blaNDM–1 and mcr-9 in a Conjugative IncHI2/HI2A Plasmid From a Bloodstream Infection-Causing Carbapenem-Resistant Klebsiella pneumoniae
Authors: Liu Zhou , Hang Xiubing , Xiao Xiao , Chu Wenwen , Li Xin , Liu Yangyang , Li Xi , Zhou Qiang , Li Jiabin
Published: December 2021

Spread of the carbapenemase-encoding and mobilized colistin resistance (mcr) genes among Enterobacteriales poses a great threat to global public health, especially when the both genes are transferred by a single plasmid. Here, we identified a blaNDM–1- and mcr-9-co-encoding plasmid harbored by a clinical isolate of Klebsiella pneumoniae (KPN710429). KPN710429 was recovered from a blood sample from an inpatient in a tertiary hospital in China, and antimicrobial susceptibility testing showed that it was multidrug-resistant and only susceptible to aztreonam, colistin, and tigecycline. KPN710429 belongs to sequence type (ST) 1308 and capsular serotype KL144. The string test of KPN710429 was negative, and this strain didn’t exhibit a hypervirulent phenotype according to serum-killing and Galleria mellonella lethality assessments. Whole-genome sequencing revealed the KPN710429 genome comprises a single chromosome and three plasmids. All virulence associated genes were harbored by chromosome. Most of its antimicrobial resistance genes, including blaNDM–1 and mcr-9 were carried by plasmid pK701429_2, belonging to the incompatibility (Inc) HI2/HI2A group and ST1. Comparative genomics assays indicates that pK710429_2 could be a hybrid plasmid, formed by a Tn6696-like blaNDM–1 region inserting into a mcr-9-positive-IncHI2/HI2A plasmid. pK710429_2 contained the conjugative transfer gene regions, Tra1 and Tra2, with some structural variations, and conjugation assays revealed that pK710429_2 was transferable. Although pK710429_2 lacked the qseB-qseC regulatory genes, mcr-9 expression was upregulated after pretreatment with colistin for 6 h, leading to colistin resistance in KPN710429. To our knowledge, this is the first report of a blaNDM–1- and mcr-9-co-encoding transferable plasmid harbored by a bloodstream-infection-causing K. pneumoniae strain in China. Effective surveillance should be implemented to assess the prevalence of the plasmid co-harboring carbapenemase-encoding gene and mcr-9.
Tropheryma whipplei Detection by Nanopore Sequencing in Patients With Interstitial Lung Disease
Authors: Guo Yifan , Li Lijuan , Li Zhenzhong , Sun Lingxiao , Wang Hui
Published: December 2021

Tropheryma whipplei is a bacterium associated with Whipple’s disease, which commonly manifests as weight loss, arthralgia, and diarrhea. The most frequently involved organs comprise the heart and eyes, in addition to the central nervous system. Few studies have explored the relationship between T. whipplei and pneumonia. Herein, we report three patients with interstitial lung disease (ILD) of unknown cause, whose bronchoalveolar lavage fluid (BALF) were evaluated via Nanopore sequencing. In our in-house BALF Nanopore platform, human DNA was removed with saponin, to improve the reads ratio of microorganisms/host. T. whipplei was the sole or most abundant pathogen in all the patients, comprising 1,385, 826, and 285 reads. The positive result was confirmed via quantitative polymerase chain reaction (PCR) with two pairs of primers (cycle threshold value: 33.26/36.29; 31.68/32.01; 28.82/28.80) and Sanger sequencing. To our knowledge, this is the first report of T. whipplei detection using Nanopore-based sequencing. The turnaround time was approximately 6–8 h in clinical laboratories, including less than 1 h for analysis. In conclusion, the results of this study confirm that Nanopore sequencing can rapidly detect rare pathogens, to improve clinical diagnosis. In addition, diagnosis of Whipple’s disease should be combined other laboratory findings, such as periodic acid-Schiff (PAS) staining, and considered a possibility in middle-aged men presenting with ILD and a clinical history of unexplained arthralgia and/or fever.
Profiles of Small Regulatory RNAs at Different Growth Phases of Streptococcus thermophilus During pH-Controlled Batch Fermentation
Authors: Liu Gefei , Chang Haode , Qiao Yali , Huang Kai , Zhang Ao , Zhao Yu , Feng Zhen
Published: December 2021

Small regulatory RNA (sRNA) has been shown to play an important role under various stress conditions in bacteria, and it plays a vital role in regulating growth, adaptation and survival through posttranscriptional control of gene expression in bacterial cells. Streptococcus thermophilus is widely used as a starter culture in the manufacture of fermented dairy products. However, the lack of reliable information on the expression profiles and potential physiological functions of sRNAs in this species hinders our understanding of the importance of sRNAs in S. thermophilus. The present study was conducted to assess the expression profiles of sRNAs in S. thermophilus and to identify sRNAs that exhibited significant changes. A total of 530 potential sRNAs were identified, including 198 asRNAs, 135 sRNAs from intergenic regions, and 197 sRNAs from untranslated regions (UTRs). Significant changes occurred in the expression of 238, 83, 194, and 139 sRNA genes during the lag, early exponential growth, late exponential growth, and stationary phases, respectively. The expression of 14 of the identified sRNAs was verified by qRT-PCR. Predictions of the target genes of these candidate sRNAs showed that the primary metabolic pathways targeted were involved in carbon metabolism, biosynthesis of amino acids, ABC transporters, the metabolism of amino and nucleotide sugars, purine metabolism, and the phosphotransferase system. The expression of the predicted target genes was further analyzed to better understand the roles of sRNAs during different growth stages. The results suggested that these sRNAs play crucial roles by regulating biological pathways during different growth phases of S. thermophilus. According to the results, sRNAs sts141, sts392, sts318, and sts014 are involved in the regulation of osmotic stress. sRNAs sts508, sts087, sts372, sts141, sts375, and sts119 are involved in the regulation of starvation stress. sRNAs sts129, sts226, sts166, sts231, sts204, sts145, and sts236 are involved in arginine synthesis. sRNAs sts033, sts341, sts492, sts140, sts230, sts172, and sts377 are involved in the ADI pathway. The present study provided valuable information for the functional study of sRNAs in S. thermophilus and indicated a future research direction for sRNA in S. thermophilus. Overall, our results provided new insights for understanding the complex regulatory network of sRNAs in S. thermophilus.
Comparative Genomic Analyses Reveal Potential Factors Responsible for the ST6 Oxacillin-Resistant Staphylococcus lugdunensis Endemic in a Hospital
Authors: Chang Shih-Cheng , Lin Lee-Chung , Lu Jang-Jih
Published: December 2021

Oxacillin-resistant Staphylococcus lugdunensis (ORSL) is considered a life-threatening isolate in healthcare settings. Among ORSL clones, ST6-SCCmec II strains are associated with an endemic spread in hospitals. We analyzed the complete genome of ORSL CGMH-SL118, a representative strain. Results revealed that this strain contained three MGEs (two prophages and one plasmid) other than the SCCmec II element, which showed remarkable differences in genome organization compared to the reference strains from NCBI. Eight multidrug-resistant genes were identified. All but blaZ were carried by MGEs, such as the SCCmec II element [mecA, ant (9)-Ia, and ermA] and the prophage φSPbeta [aac (6')-aph (2'), aph (3')-III, and ant (6)-Ia], indicating that MGEs carrying multidrug-resistant genes may be important for ST6 strains. The prophage φSPbeta contains sasX gene, which was responsible for the pathogenesis of Staphylococcus aureus. A phage-mediated resistant island containing fusB (SlRIfusB-118) was found near φSPbeta, which was highly homologous to type III SeRIfusB-5907 of Staphylococcus epidermidis. In contrast to previous studies, over 20% of ST6 isolates showed a fusidic acid-resistant phenotype, suggesting that phage-mediated intraspecies transmission of resistant islands may become an important issue for ST6 strains. Sixty-eight clinical isolates of ST6 Staphylococcus lugdunensis (50 OSSL, oxacillin-sensitive S. lugdunensis, and 18 ORSL, including CGMH-SL118) collected from various types of specimens in the hospital were studied. Among these isolates in this study, ORSL showed similar drug-resistant genes and phenotypes as CGMH-SL118. The comparative genomic analyses highlight the contribution of MGEs in the development and dissemination of antimicrobial resistance in ST6 strains, suggesting that resistance determinants and virulence factors encoded by MGEs provide a survival advantage for successful colonization and spread in healthcare settings.
Comparative Metagenomic Analysis of Two Hot Springs From Ourense (Northwestern Spain) and Others Worldwide
Authors: DeCastro María-Eugenia , Escuder-Rodríguez Juan-José , Becerra Manuel , Rodríguez-Belmonte Esther , González-Siso María-Isabel
Published: December 2021

With their circumneutral pH and their moderate temperature (66 and 68°C, respectively), As Burgas and Muiño da Veiga are two important human-use hot springs, previously studied with traditional culture methods, but never explored with a metagenomic approach. In the present study, we have performed metagenomic sequence-based analyses to compare the taxonomic composition and functional potential of these hot springs. Proteobacteria, Deinococcus-Thermus, Firmicutes, Nitrospirae, and Aquificae are the dominant phyla in both geothermal springs, but there is a significant difference in the abundance of these phyla between As Burgas and Muiño da Veiga. Phylum Proteobacteria dominates As Burgas ecosystem while Aquificae is the most abundant phylum in Muiño da Veiga. Taxonomic and functional analyses reveal that the variability in water geochemistry might be shaping the differences in the microbial communities inhabiting these geothermal springs. The content in organic compounds of As Burgas water promotes the presence of heterotrophic populations of the genera Acidovorax and Thermus, whereas the sulfate-rich water of Muiño da Veiga favors the co-dominance of genera Sulfurihydrogenibium and Thermodesulfovibrio. Differences in ammonia concentration exert a selective pressure toward the growth of nitrogen-fixing bacteria such as Thermodesulfovibrio in Muiño da Veiga. Temperature and pH are two important factors shaping hot springs microbial communities as was determined by comparative analysis with other thermal springs.