Compositional and functional aberrance of the gut microbiota in treatment-naïve patients with primary Sjögren's syndrome.

OBJECTIVES: To investigate the compositional and functional characteristics of the gut microbiota in primary Sjögren's syndrome (pSS) and compare them with those in systemic lupus erythematosus (SLE). METHODS: Stool samples from 78 treatment-naïve pSS patients and 78 matched healthy controls were detected by shotgun metagenomic sequencing and compared with those from 49 treatment-naïve SLE patients. The virulence loads and mimotopes of the gut microbiota were also assessed by sequence alignment. RESULTS: The gut microbiota of treatment-naïve pSS patients had lower richness and evenness and showed a different community distribution than that of healthy controls. The microbial species enriched in the pSS-associated gut microbiota included Lactobacillus salivarius, Bacteroides fragilis, Ruminococcus gnavus, Clostridium bartlettii, Clostridium bolteae, Veillonella parvula, and Streptococcus parasanguinis. Lactobacillus salivarius was the most discriminating species in the pSS patients, especially in those with interstitial lung disease (ILD). Among the differentiating microbial pathways, the superpathway of l-phenylalanine biosynthesis was also further enriched in pSS complicated with ILD. There were more virulence genes carried by the gut microbiota in pSS patients, most of which encoded peritrichous flagella, fimbriae, or curli fimbriae, three types of bacterial surface organelles involved in bacterial colonization and invasion. Five microbial peptides with the potential to mimic pSS-related autoepitopes were also enriched in the pSS gut. SLE and pSS shared significant gut microbial traits, including community distribution, altered microbial taxonomy and pathways, and enriched virulence genes. However, Ruminococcus torques was depleted in pSS patients but enriched in SLE patients compared to healthy controls. CONCLUSIONS: The gut microbiota in treatment-naïve pSS patients was disturbed and shared significant similarity with that in SLE patients.

Compositional and functional aberrance of the gut microbiota in treatment naïve patients with primary Sjögren's syndrome.

OBJECTIVES: To investigate the compositional and functional characteristics of the gut microbiota in primary Sjögren's syndrome (pSS) and compare them with those in systemic lupus erythematosus (SLE). METHODS: Stool samples from 78 treatment naïve pSS patients and 78 matched healthy controls were detected by shotgun metagenomic sequencing and compared with those from 49 treatment naïve SLE patients. The virulence loads and mimotopes of the gut microbiota were also assessed by sequence alignment. RESULTS: The gut microbiota of treatment naïve pSS patients had lower richness and evenness and showed a different community distribution than that of healthy controls. The microbial species enriched in the pSS-associated gut microbiota included Lactobacillus salivarius, Bacteroides fragilis, Ruminococcus gnavus, Clostridium bartlettii, Clostridium bolteae, Veillonella parvula, and Streptococcus parasanguinis. Lactobacillus salivarius was the most discriminating species in the pSS patients, especially in those with interstitial lung disease (ILD). Among the differentiating microbial pathways, the superpathway of l-phenylalanine biosynthesis was also further enriched in pSS complicated with ILD. There were more virulence genes carried by the gut microbiota in pSS patients, most of which encoded peritrichous flagella, fimbriae, or curli fimbriae, three types of bacterial surface organelles involved in bacterial colonization and invasion. Five microbial peptides with the potential to mimic pSS-related autoepitopes were also enriched in the pSS gut. SLE and pSS shared significant gut microbial traits, including the community distribution, altered microbial taxonomy and pathways, and enriched virulence genes. However, Ruminococcus torques was depleted in pSS patients but enriched in SLE patients compared to that in healthy controls. CONCLUSIONS: The gut microbiota in treatment naïve pSS patients was disturbed and shared significant similarity with that in SLE patients.

Genotypic differences in CC224, CC363, CC449 and CC446 of Moraxella catarrhalis isolates based on whole genome SNP, MLST and PFGE typing.

Understanding the evolutionary path of M. catarrhalis from macrolide-susceptible to macrolide-resistant organism, is important for hindering macrolide resistance from propagation. Multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole genome SNP typing (WGST), as useful and practical typing tools, have both advantages and disadvantages. We studied the utility of these 3 typing methods, including the level of agreement, consistency and drawbacks, in characterizing M. catarrhalis clones and clonal complexes. We focused on four clonal complexes [CC224, CC363, CC449 (CCN10) and CC446 (CCN08)] and found that PFGE and WGST had a high level of agreement and a proper consistency of the same clone or very closely related clones, while MLST is less discriminatory for different clones. Furthermore, we also established an evolutionary distance cut-off value for "The same clone". Moreover, we detected macrolide-resistant M. catarrhalis in CC224, which had previously been considered as a macrolide-susceptible clonal complex. A higher number of isolates belonged to ST215 compared to ST446, implying that ST215 is more likely to be the primary founder. Our study also demonstrated that all the four clonal complexes belong to the M. catarrhalis lineage 1, which is considered to be related to increased virulence potential and serum resistance. We also observed that copB II was highly related to CC449 and LOS type B was mainly confined in CC224. In conclusion, these findings provide further insight into the evolutionary characteristics of M. catarrhalis.

A-to-I RNA Editing in Klebsiella pneumoniae Regulates Quorum Sensing and Affects Cell Growth and Virulence.

Millions of adenosine (A) to inosine (I) RNA editing events are reported and well-studied in eukaryotes; however, many features and functions remain unclear in prokaryotes. By combining PacBio Sequel, Illumina whole-genome sequencing, and RNA Sequencing data of two Klebsiella pneumoniae strains with different virulence, a total of 13 RNA editing events are identified. The RNA editing event of badR is focused, which shows a significant difference in editing levels in the two K. pneumoniae strains and is predicted to be a transcription factor. A hard-coded Cys is mutated on DNA to simulate the effect of complete editing of badR. Transcriptome analysis identifies the cellular quorum sensing (QS) pathway as the most dramatic change, demonstrating the dynamic regulation of RNA editing on badR related to coordinated collective behavior. Indeed, a significant difference in autoinducer 2 activity and cell growth is detected when the cells reach the stationary phase. Additionally, the mutant strain shows significantly lower virulence than the WT strain in the Galleria mellonella infection model. Furthermore, RNA editing regulation of badR is highly conserved across K. pneumoniae strains. Overall, this work provides new insights into posttranscriptional regulation in bacteria.

Correlation of Moraxella catarrhalis macrolide susceptibility with the ability to adhere and invade human respiratory epithelial cells.

Recently, the prevalence of macrolide-resistant Moraxella catarrhalis has been reported, especially among Chinese children. The fitness cost of resistance is reported to render the resistant bacteria less virulent. To investigate the correlation between macrolide susceptibility of M. catarrhalis and pathogenicity, the whole genome of 70 M. catarrhalis isolates belonging to four clonal complexes with different macrolide susceptibilities was sequenced. The gene products were annotated with the Gene Ontology terms. Based on 46 extracted essential virulence genes, 19 representative isolates were selected to infect type II alveolar cells (A549 cells). The ability of these isolates to adhere and invade human epithelial cells and to produce cytokines was comparatively analysed. Furthermore, mice were infected with a pair of M. catarrhalis isolates with different pathogenic behaviours and macrolide susceptibilities to examine pulmonary clearance, histological findings, and the production of cytokines. The percentages of annotations for binding, metabolic process, cellular process, and cell were non-significantly different between the macrolide-resistant and macrolide-susceptible groups. The presence of uspA2, uspA2H, pilO, lbpB, lex1, modM, mboIA, and mboIB significantly differed among the four clonal complexes and macrolide susceptibility groups. Furthermore, compared with those in macrolide-susceptible isolates, the adhesion ability was stronger (P = 0.0019) and the invasion ability was weaker (P < 0.0001) in the macrolide-resistant isolates. Mouse experiments revealed that pulmonary macrophages elicit immune responses against M. catarrhalis infection by significantly upregulating the Csf2, Il4, Il13, Il1b, Il6, Tnf, and Il18. Therefore, M. catarrhalis populations exhibited diverse pathogenicity in vitro and in vivo.

First two fungemia cases caused by Candida haemulonii var. vulnera in China with emerged antifungal resistance.

Candida haemulonii var. vulnera is a rare variant of C. haemulonii, which has been previously reported to cause human infections. Owing to the close kinship between C. haemulonii sensu stricto and C. haemulonii var. vulnera, accurate identification of C. haemulonii var. vulnera relied on DNA sequencing assay targeting, for example, rDNA internal transcribed spacer (ITS) region. In this work, two strains of C. haemulonii var. vulnera were collected from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). The identification capacity of three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and VITEK 2 YST ID biochemical methods were evaluated against ITS sequencing. In addition, antifungal susceptibility testing was performed using Sensititre YeastOne. Moreover, we comprehensively screened drug-resistant related genes by whole-genome sequencing. The two strains were not correctly identified to species variant level using MALDI-TOF MS and YST ID cards. Both strains were resistant to amphotericin B (minimum inhibitory concentration [MIC] > 2 µg/ml). Moreover, strain F4564 and F4584 exhibited high MIC to fluconazole (>256 µg/ml) and 5-flucytosine (>64 µg/ml), respectively, which were supposed to result from key amino acid substitutions Y132F and G307A in Erg11p and V58fs and G60K substitutions in Fur1p. The rare species C. haemulonii var. vulnera has emerged in China, and such drug-resistant fungal species that can cause invasive diseases require further close attention.

Antifungal susceptibility profiles and drug resistance mechanisms of clinical Candida duobushaemulonii isolates from China.

Candida duobushaemulonii, type II Candida haemulonii complex, is closely related to Candida auris and capable of causing invasive and non-invasive infections in humans. Eleven strains of C. duobushaemulonii were collected from China Hospital Invasive Fungal Surveillance Net (CHIF-NET) and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), VITEK 2 Yeast Identification Card (YST), and internal transcribed spacer (ITS) sequencing. Whole genome sequencing of C. duobushaemulonii was done to determine their genotypes. Furthermore, C. duobushaemulonii strains were tested by Sensititre YeastOne™ and Clinical and Laboratory Institute (CLSI) broth microdilution panel for antifungal susceptibility. Three C. duobushaemulonii could not be identified by VITEK 2. All 11 isolates had high minimum inhibitory concentrations (MICs) to amphotericin B more than 2 µg/ml. One isolate showed a high MIC value of ≥64 µg/ml to 5-flucytosine. All isolates were wild type (WT) for triazoles and echinocandins. FUR1 variation may result in C. duobushaemulonii with high MIC to 5-flucytosine. Candida duobushaemulonii mainly infects patients with weakened immunity, and the amphotericin B resistance of these isolates might represent a challenge to clinical treatment.

Clinical-radiological characteristics and intestinal microbiota in patients with pancreatic immune-related adverse events.

BACKGROUND: The pancreatic immune-related adverse event (irAE) is a rare but increasingly occurrence disease with limited knowledge, which was associated with the use of immune checkpoint inhibitors (ICIs). METHODS: In this case series study of pancreatic irAE patients, clinical and radiological manifestations are summarized. Baseline and post-treatment fecal microbiota of immune-related acute pancreatitis (irAP) patients were analyzed by the 16 s rDNA amplicon sequencing method. RESULTS: A total of six patients were enrolled into the study, and the onset of pancreatic irAEs occurred a median of 105 days after a median of 4.5 cycles with immune checkpoint inhibitors (ICIs). All patients had an effective response to ICIs. Abdominal pain was the main clinical manifestation. Serum amylase (sAMY) and lipase (sLIP) had dynamic changes parallel to clinical severity. Contrast-enhanced computed tomography (CT) did not accurately reveal the level of inflammation. However, magnetic resonance imaging (MRI) was a sensitive imaging method which showed decreased and increased signal intensity of pancreatic parenchyma in T1-weighted fat-saturated and diffusion-weighted imaging, respectively. Glucocorticoids were the main treatment with a rapid initial effect followed by a slow improvement. After reinitiation of ICI therapy, pancreatic irAEs either deteriorated, remained stable or the patient developed severe pancreatic ß-cell destruction without irAP recurrence. The baseline microbiota of irAP had low Bacteroidetes/Firmicutes ratio at phylum level, low relative abundance of Alistipes, Bacteroides and high Lachnospiraceae at genus level, compared to levels of pancreatic ß-cell destruction and post-treatment of irAP. CONCLUSIONS: Pancreatic irAE patients had corresponding abdominal pain and increase in sAMY/sLIP. MRI was found to be an ideal imaging modality. Treatment with glucocorticoids were the main approach. The microbiota showed relative changes at baseline and during treatment.

Species distribution and antifungal susceptibilities of clinical isolates of Penicillium and Talaromyces species in China.

Morphologically identified Penicillium (n = 103) and Talaromyces marneffei (n = 8) isolates were collected from various clinical sources between 2016 and 2017 at a medical centre in Beijing, China. Identification to species level was confirmed by sequencing of the internal transcribed spacer (ITS) region, ß-tubulin gene (benA) and RNA polymerase II second largest subunit (RPB2) gene. Of the 111 isolates, 56 (50.5%) were identified as Penicillium spp. and 55 (49.5%) as Talaromyces spp. Eleven species of Penicillium were detected, of which Penicillium oxalicum was the commonest, accounting for 51.8% (29/56), followed by Penicillium rubens (10.7%; 6/56) and Penicillium citrinum (10.7%; 6/56). Among the 55 Talaromyces isolates, nine species were identified, with Talaromyces funiculosus (36.4%; 20/55), Talaromyces stollii (27.3%; 15/55) and Talaromyces marneffei (14.5%; 8/55) being the most common. Of note, 89.3% (50/56) of the Penicillium isolates and 98.2% (54/55) of the Talaromyces isolates exhibited growth at 37°C. The isolates were mainly recovered from patients with pulmonary disorders (56.8%; 63/111), autoimmune disease (12.6%; 14/111) and AIDS (5.4%; 6/111). The azoles and amphotericin B exhibited potent activity against T. marneffei, while various levels of activity were observed against Penicillium and other Talaromyces species The echinocandins had the lowest MECs (MEC90, ≤0.12 mg/L) against most Penicillium and Talaromyces species, with the exception of T. marneffei whose MEC90 (4 mg/L) was five or more dilutions higher than that of the other species tested. These data on the species distribution and antifungal susceptibility expand the current clinical knowledge of Penicillium and Talaromyces species.

An Autoimmunogenic and Proinflammatory Profile Defined by the Gut Microbiota of Patients With Untreated Systemic Lupus Erythematosus.

OBJECTIVE: Changes in gut microbiota have been linked to systemic lupus erythematosus (SLE), but knowledge is limited. Our study aimed to provide an in-depth understanding of the contribution of gut microbiota to the immunopathogenesis of SLE. METHODS: Fecal metagenomes from 117 patients with untreated SLE and 52 SLE patients posttreatment were aligned with 115 matched healthy controls and analyzed by whole-genome profiling. For comparison, we assessed the fecal metagenome of MRL/lpr mice. The oral microbiota origin of the gut species that existed in SLE patients was documented by single-nucleotide polymorphism-based strain-level analyses. Functional validation assays were performed to demonstrate the molecular mimicry of newly found microbial peptides. RESULTS: Gut microbiota from individuals with SLE displayed significant differences in microbial composition and function compared to healthy controls. Certain species, including the Clostridium species ATCC BAA-442 as well as Atopobium rimae, Shuttleworthia satelles, Actinomyces massiliensis, Bacteroides fragilis, and Clostridium leptum, were enriched in SLE gut microbiota and reduced after treatment. Enhanced lipopolysaccharide biosynthesis aligned with reduced branched chain amino acid biosynthesis was observed in the gut of SLE patients. The findings in mice were consistent with our findings in human subjects. Interestingly, some species with an oral microbiota origin were enriched in the gut of SLE patients. Functional validation assays demonstrated the proinflammatory capacities of some microbial peptides derived from SLE-enriched species. CONCLUSION: This study provides detailed information on the microbiota of untreated patients with SLE, including their functional signatures, similarities with murine counterparts, oral origin, and the definition of autoantigen-mimicking peptides. Our data demonstrate that microbiome-altering approaches may offer valuable adjuvant therapies in SLE.