Antibiotic resistance of Mycoplasma Synoviae strains isolated in China from 2016 to 2019

Background In the past decade, Mycoplasma synoviae (M. synoviae) infection has become widely prevalent in China, has caused serious economic losses and has become one of the most important diseases in the chicken industry. Medication is a general approach for the control of M. synoviae infection, but antibiotics are sometimes ineffective in clinical practice. To investigate the sensitivity of M. synoviae to antimicrobials commonly used in the treatment of M. synoviae infection, the antibiotic susceptibility of 32 M. synoviae strains isolated from China from 2016 to 2019 were determined using the minimum inhibitory concentration (MIC) method. Results All isolates had low MIC values for the combination of lincomycin and spectinomycin, pleuromutilin, and macrolides. However, the M. synoviae isolates displayed variance in MICs for doxycycline hydrochloride with a range of 0.25 to 8 μg/mL, and oxytetracycline hydrochloride with a range of 0.5 to 8 μg/mL. Three and one M. synoviae isolates showed intermediate MIC values to doxycycline hydrochloride and oxytetracycline hydrochloride, respectively. High MIC values for enrofloxacin were detected in all isolates with MICs ranging from 4 to 32 μg/mL. Furthermore, comparison of the parC QRDR identified a mutation at nucleotide position 254 (C254T) resulting in a Thr 85 Ile amino acid change in all M. synoviae isolates and the reference strain ATCC 25204 being resistant to enrofloxacin. Moreover, mutations at Glu 804 Gly and Thr 686 Ala of gyrA QRDR were identified in all M. synoviae isolates and ATCC 25204. The mutation in the QRDR of the parE gene resulted in amino acid changes at positions 197 (Pro to Ser) in 27/32 M. synoviae isolates. Conclusion Three nonsynonymous mutations in gyrA and parE were first identified to be related to enrofloxacin resistance. Our results showed that M. synoviae resistance to enrofloxacin is widespread.

Exome-wide analysis of copy number variation shows association of the human leukocyte antigen region with asthma in UK Biobank

Background: The role of copy number variants (CNVs) in susceptibility to asthma is not well understood. This is, in part, due to the difficulty of accurately measuring CNVs in large enough sample sizes to detect associations. The recent availability of whole-exome sequencing (WES) in large biobank studies provides an unprecedented opportunity to study the role of CNVs in asthma. Methods: We called common CNVs in 49,953 individuals in the first release of UK Biobank WES using ClinCNV software. CNVs were tested for association with asthma in a stage 1 analysis comprising 7,098 asthma cases and 36,578 controls from the first release of sequencing data. Nominally-associated CNVs were then meta-analysed in stage 2 with an additional 17,280 asthma cases and 115,562 controls from the second release of UK Biobank exome sequencing, followed by validation and fine-mapping. Results: Five of 189 CNVs were associated with asthma in stage 2, including a deletion overlapping the HLA-DQA1 and HLA-DQB1 genes, a duplication of CHROMR/PRKRA, deletions within MUC22 and TAP2, and a duplication in FBRSL1. The HLA-DQA1, HLA-DQB1, MUC22 and TAP2 genes all reside within the human leukocyte antigen (HLA) region on chromosome 6. In silico analyses demonstrated that the deletion overlapping HLA-DQA1 and HLA-DQB1 is likely to be an artefact arising from under-mapping of reads from non-reference HLA haplotypes, and that the CHROMR/PRKRA and FBRSL1 duplications represent presence/absence of pseudogenes within the HLA region. Bayesian fine-mapping of the HLA region suggested that there are two independent asthma association signals. The variants with the largest posterior inclusion probability in the two credible sets were an amino acid change in HLA-DQB1 (glutamine to histidine at residue 253) and a multi-allelic amino acid change in HLA-DRB1 (presence/absence of serine, glycine or leucine at residue 11). Conclusions: At least two independent loci characterised by amino acid changes in the HLA-DQA1, HLA-DQB1 and HLA-DRB1 genes are likely to account for association of SNPs and CNVs in this region with asthma. The high divergence of haplotypes in the HLA can give rise to spurious CNVs, providing an important, cautionary tale for future large-scale analyses of sequencing data.

Estimating the transmission advantage of the D614G mutant strain of SARS-CoV-2, December 2019 to June 2020

Introduction The SARS-CoV-2 lineages carrying the amino acid change D614G have become the dominant variants in the global COVID-19 pandemic. By June 2021, all the emerging variants of concern carried the D614G mutation. The rapid spread of the G614 mutant suggests that it may have a transmission advantage over the D614 wildtype. Aim Our objective was to estimate the transmission advantage of D614G by integrating phylogenetic and epidemiological analysis. Methods We assume that the mutation D614G was the only site of interest which characterised the two cocirculating virus strains by June 2020, but their differential transmissibility might be attributable to a combination of D614G and other mutations. We define the fitness of G614 as the ratio of the basic reproduction number of the strain with G614 to the strain with D614 and applied an epidemiological framework for fitness inference to analyse SARS-CoV-2 surveillance and sequence data. Results Using this framework, we estimated that the G614 mutant is 31% (95% credible interval: 28–34) more transmissible than the D614 wildtype. Therefore, interventions that were previously effective in containing or mitigating the D614 wildtype (e.g. in China, Vietnam and Thailand) may be less effective against the G614 mutant. Conclusion Our framework can be readily integrated into current SARS-CoV-2 surveillance to monitor the emergence and fitness of mutant strains such that pandemic surveillance, disease control and development of treatment and vaccines can be adjusted dynamically.

Dramatic activation of an antibody by a single amino acid change in framework

Antibody function is typically entirely dictated by the Complementarity Determining Regions (CDRs) that directly bind to the antigen, while the framework region acts as a scaffold for the CDRs and maintains overall structure of the variable domain. We recently reported that the rabbit monoclonal antibody 4A11 (rbt4A11) disrupts signaling through both TGFβ2 and TGFβ3 (Sun et al. in Sci Transl Med, 2021. https://doi.org/10.1126/scitranslmed.abe0407). Here, we report a dramatic, unexpected discovery during the humanization of rbt4A11 where, two variants of humanized 4A11 (h4A11), v2 and v7 had identical CDRs, maintained high affinity binding to TGFβ2/3, yet exhibited distinct differences in activity. While h4A11.v7 completely inhibited TGFβ2/3 signaling like rbt4A11, h4A11.v2 did not. We solved crystal structures of TGFβ2 complexed with Fab fragments of h4A11.v2 or h4A11.v7 and identified a novel interaction between the two heavy chain molecules in the 2:2 TGFb2:h4A11.v2-Fab complex. Further characterization revealed that framework residue variations at either position 19, 79 or 81 (Kabat numbering) of the heavy chain strikingly converts h4A11.v2 into an inhibitory antibody. Our work suggests that in addition to CDRs, framework residues and interactions between Fabs in an antibody could be engineered to further modulate activity of antibodies.

Dramatic Activation of an Antibody by a Single Amino Acid Change in Framework

Antibody function is typically entirely dictated by the Complementarity Determining Regions (CDRs) that directly bind to the antigen, while the framework region acts as a scaffold for the CDRs and maintains overall structure of the variable domain. We recently reported that the rabbit monoclonal antibody 4A11 (rbt4A11) disrupts signaling through both TGFβ2 and TGFβ31. Here, we report a dramatic, unexpected discovery during the humanization of rbt4A11 where, two variants of humanized 4A11 (h4A11), v2 and v7 had identical CDRs, maintained high affinity binding to TGFβ2/3, yet exhibited distinct differences in activity. While h4A11.v7 completely inhibited TGFβ2/3 signaling like rbt4A11, h4A11.v2 did not. We solved crystal structures of TGFβ2 complexed with Fab fragments of h4A11.v2 or h4A11.v7 and identified a novel interaction between the two heavy chain molecules in the 2:2 TGFb2:h4A11.v2-Fab complex. Further characterization revealed that framework residue variations at either position 19, 79 or 81 of the heavy chain strikingly converts h4A11.v2 into an inhibitory antibody. Our work suggests that in addition to CDRs, framework residues and interactions between Fabs in an antibody could be engineered to further modulate activity of antibodies.

Molecular Characterization of Fluoroquinolone-Resistant Bartonella bacilliformis

The presence of amino acid changes in GyrA, GyrB, ParC, ParE, and in a proposed chromosomal chloramphenicol acetyl transferase (CAT), as well as mutations at 23S rRNA, were established by PCR and sequencing in 38 B. bacilliformis clinical isolates from four different areas in Peru. Eighteen out of 24 (75%) isolates showing ciprofloxacin resistance for both disk-diffusion and e-test presented amino acid substitutions in GyrA (G89C, six isolates, A91V, 1 isolate) GyrB (S474F, 10 isolates) or both (GyrA D95N and GyrB S474F, one isolate). Two out of 14 susceptible isolates presented amino acid substitutions at GyrB (S474F) or a double substitution GyrA D95N and GyrB S474F. Of note, ciprofloxacin-resistant isolates were recovered in the four areas studied. No amino acid change was observed at ParC or ParE. Only one isolate showed chloramphenicol resistance, but no alteration was present in either 23S rRNA or CAT. B. bacilliformis resistant to quinolones are extended throughout Peru, with amino acid substitutions at GyrA or GyrB as the main, albeit not exclusive, cause. B. bacilliformis seems to have an apparent facility to develop mutations on GyrB outside the classical positions 91, 95 of GyrA and 85, 88 of ParC.

Comparative transcriptomic analysis reveals the regulatory mechanism of the gibberellic acid pathway of Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) dwarf mutants

BackgroundTartary buckwheat is an important minor crop species with high nutritional and medicinal value and is widely planted worldwide. Cultivated Tartary buckwheat plants are tall and have hollow stems that lodge easily, which severely affects their yield and hinders the development of the Tartary buckwheat industry.MethodsHeifeng No. 1 seeds were treated with ethylmethanesulfonate (EMS) to generate a mutant library. The dwarf mutantftdmwas selected from the mutagenized population, and the agronomic characteristics giving rise to the dwarf phenotype were evaluated. Ultra-fast liquid chromatography-electrospray ionization tandem mass spectrometry (UFLC-ESI–MS/MS) was performed to determine the factors underlying the different phenotypes between the wild-type (WT) andftdmplants. In addition, RNA sequencing (RNA-seq) was performed via the HiSeq 2000 platform, and the resulting transcriptomic data were analysed to identify differentially expressed genes (DEGs). Single-nucleotide polymorphism (SNP) variant analysis revealed possible sites associated with dwarfism. The expression levels of the potential DEGs between the WT andftdmmutant were then measured via qRT-PCR and fragments per kilobase of transcript per million mapped reads (FPKM).ResultThe plant height (PH) of theftdmmutant decreased to 42% of that of the WT, and compared with the WT, the mutant and had a higher breaking force (BF) and lower lodging index (LI). Lower GA4 and GA7 contents and higher contents of jasmonic acid (JA), salicylic acid (SA) and brassinolactone (BR) were detected in the stems of theftdmmutant compared with the WT. Exogenous application of GAs could not revert the dwarfism of theftdmmutant. On the basis of the transcriptomic analysis, 146 homozygous SNP loci were identified. In total, 12 DEGs with nonsynonymous mutations were ultimately identified, which were considered potential candidate genes related to the dwarf trait. When the sequences of eight genes whose expression was downregulated and four genes whose expression was upregulated were compared, SKIP14, an F-box protein whose sequence is 85% homologous to that of SLY1 in Arabidopsis, presented an amino acid change (from Ser to Asn) and was expressed at a lower level in the stems of theftdmmutant compared with the WT. Hence, we speculated that this amino acid change in SKIP14 resulted in a disruption in GA signal transduction, indirectly decreasing the GA content and downregulating the expression of genes involved in GA biosynthesis or the GA response. Further studies are needed to determine the molecular basis underlying the dwarf phenotype of theftdmmutant.ConclusionWe report a Tartary buckwheat EMS dwarf mutant,ftdm, suitable for high-density planting and commercial farming. A significant decrease in GA4 and GA7 levels was detected in theftdmmutant, and 12 DEGs expressed in the stems of theftdmmutant were selected as candidates of the dwarfing gene. One nonsynonymous mutation was detected in theSKIP14gene in theftdmmutant, and this gene had a lower transcript level compared with that in the WT.

Antimicrobial Susceptibility and Genomic Analysis of Aliarcobacter cibarius and Aliarcobacter thereius, Two Rarely Detected Aliarcobacter Species

Aliarcobacter cibarius and Aliarcobacter thereius are two rarely detected Aliarcobacter species. In the study, we analyzed the antimicrobial susceptibility and provide detailed insights into the genotype and phylogeny of both species using whole-genome sequencing. Thermophilic Campylobacter species are the most common bacterial foodborne pathogens causing gastroenteritis in humans worldwide. The genus Aliarcobacter is part of the Campylobacteraceae family and includes the species Aliarcobacter butzleri, Aliarcobacter cryaerophilus, Aliarcobacter skirrowii, and the rarely described Aliarcobacter cibarius, Aliarcobacter faecis, Aliarcobacter lanthieri, Aliarcobacter thereius, and Acrobarter trophiarum. Aliarcobacter are emergent enteropathogens and potential zoonotic agents. Here, we generated, analyzed, and characterized whole-genome sequences of Aliarcobacter cibarius and Aliarcobacter thereius. They were isolated from water poultry farms in Germany, cultured and identified by MALDI-TOF MS. With PCR the identity was verified. Antibiotic susceptibility testing was carried out with erythromycin, ciprofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, ampicillin, and cefotaxime using the gradient strip method (E-test). Whole-genome sequences were generated including those of reference strains. Complete genomes for six selected strains are reported. These provide detailed insights into the genotype. With these, we predicted in silico known AMR genes, virulence-associated genes, and plasmid replicons. Phenotypic analysis of resistance showed differences between the presence of resistance genes and the prediction of phenotypic resistance profiles. In Aliarcobacter butzleri, the nucleotide sequence of the gyrA gene (DQ464331) can show a signature mutation resulting in an amino acid change T85>I. Acrobarter cibarius and Acrobarter thereius showed the same gene as assessed by similarity annotation of the mutations 254C>G. Most of the isolates were found to be sensitive to ciprofloxacin. The ciprofloxacin-resistant Aliarcobacter thereius isolate was associated with the amino acid change T85>I. But this was not predicted with antibiotic resistance databases, before. Ultimately, a phylogenetic analysis was done to facilitate in future outbreak analysis.

A Single Amino Acid Change In Histone Methyltransferase CURLY LEAF Results In Premature Bolting In Chinese Cabbage (Brassica Rapa L. Ssp. Pekinensis)

Background: Flowering is an important inflection point in the transformation from vegetative to reproductive growth, and premature bolting severely decreases crop yield and quality. Results: In this study, a stable early-bolting mutant, ebm3, was identified in an ethyl methanesulfonate (EMS)-mutagenized population of a Chinese cabbage doubled haploid (DH) line ‘FT’. Compared with ‘FT’, ebm3 showed early bolting under natural cultivation in autumn, and curled leaves. Genetic analysis showed that the early-bolting phenotype was controlled by a single recessive nuclear gene. Modified MutMap and genotyping analyses revealed that Brebm3 (BraA04g017190.3C), encoding the histone methyltransferase CURLY LEAF (CLF), was the causal gene of the emb3. A C to T base substitution in the 14th exon of Brebm3 resulted in an amino acid change (S to F) and the early-bolting phenotype of emb3. The mutation occurred in the SET domain (Suppressor of protein-effect variegation 3-9, Enhancer-of-zeste, Trithorax), which catalyzes site- and state-specific lysine methylation in histones. Tissue-specific expression analysis showed that Brebm3 was highly expressed in the flower and bud. Promoter activity assay confirmed that Brebm3 promoter was active in inflorescences. Subcellular localization analysis revealed that Brebm3 localized in the nucleus. Transcriptomic studies supported that Brebm3 mutation might repress H3K27me3 deposition and activate expression of the AGAMOUS (AG) and AGAMOUS-like (AGL) loci, resulting in early flowering.Conclusions: Our study revealed that an EMS-induced early-bolting mutant ebm3 in Chinese cabbage was caused by a nonsynonymous mutation in BraA04g017190.3C, encoding the histone methyltransferase CLF.These results improve our knowledge of the genetic and genomic resources of bolting and flowering, and may be beneficial to the genetic improvement of Chinese cabbage.