Comparative genomics of a novel Erwinia species associated with the Highland midge (Culicoides impunctatus).

Erwinia (Enterobacterales: Erwiniaceae) are a group of cosmopolitan bacteria best known as the causative agents of various plant diseases. However, other species in this genus have been found to play important roles as insect endosymbionts supplementing the diet of their hosts. Here, I describe Candidatus Erwinia impunctatus (Erwimp) associated with the Highland midge Culicoides impunctatus (Diptera: Ceratopogonidae), an abundant biting pest in the Scottish Highlands. The genome of this new Erwinia species was assembled using hybrid long and short read techniques, and a comparative analysis was undertaken with other members of the genus to understand its potential ecological niche and impact. Genome composition analysis revealed that Erwimp is similar to other endophytic and ectophytic species in the genus and is unlikely to be restricted to its insect host. Evidence for an additional plant host includes the presence of a carotenoid synthesis operon implicated as a virulence factor in plant-associated members in the sister genus Pantoea. Unique features of Erwimp include several copies of intimin-like proteins which, along with signs of genome pseudogenization and a loss of certain metabolic pathways, suggests an element of host restriction seen elsewhere in the genus. Furthermore, a screening of individuals over two field seasons revealed the absence of the bacteria in Culicoides impunctatus during the second year indicating this microbe-insect interaction is likely to be transient. These data suggest that Culicoides impunctatus may have an important role to play beyond a biting nuisance, as an insect vector transmitting Erwimp alongside any conferred impacts to surrounding biota.

Desensitization using pegaspargase in the era of commercially available Erwinia: A single-institution report on efficacy, cost, and resource utilization.

BACKGROUND: Pegaspargase is a therapeutic enzyme that is utilized in treatment regimens targeting pediatric acute lymphoblastic leukemia. However, many patients experience hypersensitivity reactions, requiring discontinuation of the therapy. Historically, this necessitated switching to an alternative form of the drug, most commonly asparaginase Erwinia chrysanthemi; however, in recent years this was difficult due to drug shortages and eventually commercial discontinuation. We report here our experience performing pegaspargase desensitizations in patients with prior hypersensitivity reactions. PROCEDURE: Patients with a clinical hypersensitivity reaction to pegaspargase were identified. When due for their next dose, patients were admitted to the pediatric intensive care unit, bone marrow transplant unit, or oncology unit, and underwent desensitization utilizing a rigorous premedication and multistep dilution-based protocol. Serum asparaginase activity levels were drawn after desensitization to assess for therapeutic levels of enzyme activity. RESULTS: We identified 11 patients who underwent a total of 33 desensitizations to pegaspargase and calaspargase pegol-mknl. No patients experienced clinically significant hypersensitivity reactions necessitating stopping the infusion, nor administration of rescue medications. All serum asparaginase activity levels collected demonstrated enzyme activity levels above predefined therapeutic thresholds. Cost analysis revealed substantial savings when patients received asparaginase desensitization over the now commercially available asparaginase E. chrysanthemi (recombinant) rywn. CONCLUSIONS: Performing desensitization to pegaspargase in the pediatric acute lymphoblastic leukemia population is feasible, safe, and effective. It is financially advantageous over available alternative approaches, and requires fewer injections and presentations to care.

New Insights and Approach Toward the Genetic Diversity and Strain Typing of Erwinia pyrifoliae Based on rsxC, an Electron Transport Gene.

Erwinia pyrifoliae, a causal agent of black shoot blight in apple and pear trees, is a plant pathogenic bacterium first reported in South Korea. The symptoms of black shoot blight are very similar to those of the fire blight disease in apple and pear trees caused by E. amylovora, as E. pyrifoliae has a genetically very close relationship with E. amylovora. Recently, there have been reports that E. pyrifoliae causes disease in European strawberries, resulting in severe fruit loss that aroused great concern about its spread, distribution, and host range. Therefore, it is essential to establish a trustworthy approach to understanding the distribution patterns of E. pyrifoliae based on the genetic background to strengthen the barrier of potential spreading risks, although advanced methods have been provided to accurately detect E. pyrifoliae and E. amylovora. Consequently, this study discovered a noble and noteworthy gene, rsxC, capable of providing the pathogen genotype by comparing E. pyrifoliae genomic sequences in the international representative genome archive. Different numbers of 40-unit amino acid repeats in this gene among the strains induced intraspecific traits in RsxC. By comparing their repeat pattern, E. pyrifoliae isolates were divided into two main groups, branching into several clades via sequence alignment of 35 E. pyrifoliae isolates from various apple orchards from 2020 to 2021 in South Korea. The newly discovered quadraginta amino acid repeat within this gene would be a valuable genetic touchstone for determining the genotype and distribution pattern of E. pyrifoliae strains, ultimately leading to exploring their evolution. The function of amino acid repeats and the biological significance of strains need to be elucidated further.

Determination and Kinetic Characterization of a New Potential Inhibitor for AmsI Protein Tyrosine Phosphatase from the Apple Pathogen Erwinia amylovora.

Erwinia amylovora is a Gram-negative bacterium, responsible for the fire blight disease in Rosaceae plants. Its virulence is correlated with the production of an exopolysaccharide (EPS) called amylovoran, which protects the bacterium from the surrounding environment and helps its diffusion inside the host. Amylovoran biosynthesis relies on the expression of twelve genes clustered in the ams operon. One of these genes, amsI, encodes for a Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) called EaAmsI, which plays a key role in the regulation of the EPS production pathway. For this reason, EaAmsI was chosen in this work as a target for the development of new antibacterial agents against E. amylovora. To achieve this aim, a set of programs (DOCK6, OpenEye FRED) was selected to perform a virtual screening using a database of ca. 700 molecules. The six best-scoring compounds identified were tested in in vitro assays. A complete inhibition kinetic characterization carried out on the most promising molecule (n-Heptyl ß-D-glucopyranoside, N7G) showed an inhibition constant of 7.8 ± 0.6 µM. This study represents an initial step towards the development of new EaAmsI inhibitors able to act as antibacterial agents against E. amylovora infections.

Novel approach toward the understanding of genetic diversity based on the two types of amino acid repeats in Erwinia amylovora.

Erwinia amylovora is a notorious plant pathogenic bacterium of global concern that has devastated the apple and pear production industry worldwide. Nevertheless, the approaches available currently to understand the genetic diversity of E. amylovora remain unsatisfactory because of the lack of a trustworthy index and data covering the globally occurring E. amylovora strains; thus, their origin and distribution pattern remains ambiguous. Therefore, there is a growing need for robust approaches for obtaining this information via the comparison of the genomic structure of Amygdaloideae-infecting strains to understand their genetic diversity and distribution. Here, the whole-genome sequences of 245 E. amylovora strains available from the NCBI database were compared to identify intraspecific genes for use as an improved index for the simple classification of E. amylovora strains regarding their distribution. Finally, we discovered two kinds of strain-typing protein-encoding genes, i.e., the SAM-dependent methyltransferase and electron transport complex subunit RsxC. Interestingly, both of these proteins carried an amino acid repeat in these strains: SAM-dependent methyltransferase comprised a single-amino-acid repeat (asparagine), whereas RsxC carried a 40-amino-acid repeat, which was differentially distributed among the strains. These noteworthy findings and approaches may enable the exploration of the genetic diversity of E. amylovora from a global perspective.

Structure-Function Characterisation of Eop1 Effectors from the Erwinia-Pantoea Clade Reveals They May Acetylate Their Defence Target through a Catalytic Dyad.

The YopJ group of acetylating effectors from phytopathogens of the genera Pseudomonas and Ralstonia have been widely studied to understand how they modify and suppress their host defence targets. In contrast, studies on a related group of effectors, the Eop1 group, lag far behind. Members of the Eop1 group are widely present in the Erwinia-Pantoea clade of Gram-negative bacteria, which contains phytopathogens, non-pathogens and potential biocontrol agents, implying that they may play an important role in agroecological or pathological adaptations. The lack of research in this group of YopJ effectors has left a significant knowledge gap in their functioning and role. For the first time, we perform a comparative analysis combining AlphaFold modelling, in planta transient expressions and targeted mutational analyses of the Eop1 group effectors from the Erwinia-Pantoea clade, to help elucidate their likely activity and mechanism(s). This integrated study revealed several new findings, including putative binding sites for inositol hexakisphosphate and acetyl coenzyme A and newly postulated target-binding domains, and raises questions about whether these effectors function through a catalytic triad mechanism. The results imply that some Eop1s may use a catalytic dyad acetylation mechanism that we found could be promoted by the electronegative environment around the active site.

Improved production of andrimid in Erwinia persicina BST187 strain by fermentation optimization.

BACKGROUND: Andrimid is reported to be a novel kind of polyketide-nonribosomal peptide hybrid product (PK-NRPs) that inhibits fatty acid biosynthesis in bacteria. Considering its great potential in biomedicine and biofarming, intensive studies have been conducted to increase the production of andrimid to overcome the excessive costs of chemosynthesis. In screening for species with broad-spectrum antibacterial activity, we detected andrimid in the fermentation products of Erwinia persicina BST187. To increase andrimid production, the BST187 fermentation medium formulation and fermentation conditions were optimized by using systematic design of experiments (One-Factor-At-A-Time, Plackett-Burman design, Response Surface Methodology). RESULTS: The results indicate that the actual andrimid production reached 140.3 ± 1.28 mg/L under the optimized conditions (trisodium citrate dihydrate-30 g/L, beef extract-17.1 g/L, MgCl2·6H2O-100 mM, inoculation amount-1%, initial pH-7.0, fermentation time-36 h, temperature-19.7℃), which is 20-fold greater than the initial condition without optimization (7.00 ± 0.40 mg/L), consistent with the improved antibacterial effect of the fermentation supernatant. CONCLUSIONS: The present study provides valuable information for improving andrimid production via optimization of the fermentation process, which will be of great value in the future industrialization of andrimid production.

A comprehensive strategy to address shortage of Erwinia asparaginase in pediatric acute lymphoblastic leukemia.

BACKGROUND: Pegylated form of E. coli derived asparaginase (PEG) is a crucial component of pediatric ALL therapy. Patients who develop a hypersensitivity (HSR) reaction with PEG receive an alternative form - Erwinia asparaginase (EA). However, an international shortage in 2017 had made it challenging to treat these patients. We have developed a comprehensive strategy to address this need. PATIENTS AND METHODS: This is a single center, retrospective analysis. All patients receiving PEG were premedicated to reduce infusion reactions. Patients who developed HSR underwent PEG desensitization. Patients were compared to historic controls. RESULTS: Fifty-six patients were treated within the study period. There was no difference in the frequency of reactions before and after the adoption of universal premedication (p = 0.78). Eight patients (14.2%) developed either ≥ Grade 2 HSR or silent inactivation and 5 patients (62.5%) successfully underwent desensitization. The remaining three patients received EA asparaginase. This intervention led to a decrease in PEG substitution, with 3 patients (5.3%) requiring EA compared to 8 patients (15.09%) in the pre-intervention period. (p = 0.11) PEG desensitization was more cost effective than EA administration. CONCLUSION: PEG desensitization is a safe, cost effective, and practical alternative in children with ALL and a Grade 2 or higher HSR.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Examination of Large Chromosomal Inversions in the Genome of Erwinia amylovora Strains Reveals Worldwide Distribution and North America-Specific Types.

Erwinia amylovora is a relatively homogeneous species with low genetic diversity at the nucleotide level. However, phenotypic differences and genomic structural variations among E. amylovora strains have been documented. In this study, we identified 10 large chromosomal inversion (LCI) types in the Spiraeoideae-infecting (SI) E. amylovora strains by combining whole genome sequencing and PCR-based molecular markers. It was found that LCIs were mainly caused by homologous recombination events among seven rRNA operons (rrns) in SI E. amylovora strains. Although ribotyping results identified inter- and intra-variations in the internal transcribed spacer (ITS1 and ITS2) regions among rrns, LCIs tend to occur between rrns transcribed in the opposite directions and with the same tRNA content (tRNA-Glu or tRNA-Ile/Ala) in ITS1. Based on the LCI types, physical/estimated replichore imbalance (PRI/ERI) was examined and calculated. Among the 117 SI strains evaluated, the LCI types of Ea1189, CFBP1430, and Ea273 were the most common, with ERI values at 1.31, 7.87, and 4.47°, respectively. These three LCI types had worldwide distribution, whereas the remaining seven LCI types were restricted to North America (or certain regions of the United States). Our results indicated ongoing chromosomal recombination events in the SI E. amylovora population and showed that LCI events are mostly symmetrical, keeping the ERI less than 15°. These findings provide initial evidence about the prevalence of certain LCI types in E. amylovora strains, how LCI occurs, and its potential evolutionary advantage and history, which might help track the movement of the pathogen.

A novel phytopathogen Erwinia sorbitola sp. nov., isolated from the feces of ruddy shelducks.

The species in the genus Erwinia are Gram-stain-negative, facultatively anaerobic, motile, and rod-shaped. Most species in the genus Erwinia are phytopathogens. Also, Erwinia persicina was involved in several human infections. Based on the reverse microbial etiology principles, it is worth analyzing the pathogenicity of species in this genus. In this study, we isolated and sequenced two species of Erwinia. Phylogenetic, phenotypic, biochemical, and chemotaxonomic analyses were performed to identify its taxonomy position. The virulence tests on plant leaves and pear fruits were used to identify the plant pathogenicity of two species of Erwinia. Bioinformatic methods predicted the possible pathogenic determinants based on the genome sequence. Meanwhile, adhesion, invasion, and cytotoxicity assays on RAW 264.7 cells were applied to identify animal pathogenicity. We isolated two Gram-stain-negative, facultatively anaerobic, motile, and rod-shaped strains from the feces of ruddy shelducks in the Tibet Plateau of China, designated J780T and J316. Distinct phylogenetic, genomic, phenotypic, biochemical, and chemotaxonomic characters of J780T and J316 identified they were novel species and belonged to the genus Erwinia, for which the name Erwinia sorbitola sp. nov. was proposed, the type strain was J780T (= CGMCC 1.17334T = GDMCC 1.1666T = JCM 33839T). Virulence tests showed blight and rot on the leaves and pear fruits confirmed Erwinia sorbitola sp. nov. was a phytopathogen. Predicted gene clusters of motility, biofilm formation, exopolysaccharides, stress survival, siderophores, and Type VI secretion system might be the causes of pathogenicity. In addition, predicted polysaccharide biosynthesis gene clusters on the genome sequence, and the high capacity for adhesion, invasion, and cytotoxicity to animal cells confirmed it has pathogenicity on animals. In conclusion, we isolated and identified a novel phytopathogen Erwinia sorbitola sp. nov. in ruddy shelducks. A predefined pathogen is beneficial for preventing from suffering potential economic losses caused by this new pathogen.

Three novel Erwinia billingiae phages isolated from organic waste represent three new genera.

Despite the ecological significance of viral communities, phages remain insufficiently studied. Current genomic databases lack high-quality phage genome sequences linked to specific bacteria. Bacteria of the genus Erwinia are known to colonize the phyllosphere of plants, both as commensals and as pathogens. We isolated three Erwinia billingiae phages-Zoomie, Pecta, and Snitter-from organic household waste. Based on sequence similarity to their closest relatives, we propose that they represent three new genera: "Pectavirus" within the family Zobellviridae, "Snittervirus" in the subfamily Tempevirinae, family Drexlerviridae, and "Zoomievirus" within the family Autographiviridae, which, together with the genus Limelightvirus, may constitute a new subfamily.

Asparaginase activity monitoring and management of asparaginase hypersensitivity reactions in Canada.

INTRODUCTION: Pegaspargase can cause anti-asparaginase antibody formation, which can decrease its effectiveness without causing any clinically apparent reaction (silent inactivation). When a patient has silent inactivation, a switch to Erwinia anti-asparaginase is warranted, but there is currently a global shortage of Erwinia. The only way to identify silent inactivation is to measure an asparaginase level. However, routine asparaginase level monitoring is not currently standard of care at all Canadian centers. This study aims to identify variations in practice regarding asparaginase level monitoring and Erwinia use. METHODS: A 21-item survey was developed using OPINIO software and distributed to all Pediatric Hematology-Oncologists in Canada from February to October 2020. RESULTS: Respondents represented 15 hospitals across each region of Canada (response rate = 52%). Only 39.2% of respondents reported routinely measuring asparaginase levels, yet 53% of respondents have modified therapy from pegaspargase to Erwinia in up to half of their patients. The most common reason for not measuring asparaginase levels was not knowing how to use levels clinically (25.5%). There was variation in the timing of levels and their target. CONCLUSIONS: We identified substantial variation in asparaginase activity monitoring practices across Canada. Therefore, future research should aim to develop a national practice guideline on asparaginase activity monitoring.

Isolation of Streptomycin-Resistant Erwinia pyrifoliae in Korea.

As a black shoot blight disease-causing agent, Erwinia pyrifoliae was first reported in 1995 in Korea. A total of 101 isolates of E. pyrifoliae were isolated from samples showing bacterial symptoms collected from apple and pear orchards between 2020 and 2021. These isolates were screened for streptomycin resistance, with one from an orchard in Gwangju showing resistance at 100 µg/ml streptomycin. This streptomycin-resistant E. pyrifoliae (EpSmR) isolate was identified via polymerase chain reaction amplification of the strA/strB gene and an internal region of the ribosomal rpsL gene containing codon 43. EpSmR has a point mutation that altered this codon from lysine (AAA) to threonine (ACA). The strA and strB genes were not identified in EpSmR. EpSmR showed a high resistance to streptomycin (>50,000 µg/ml). This is the first study reporting EpSmR, which emerged due to a mutation in codon 43 of the rpsL gene.

Recombinant Erwinia asparaginase (JZP458) in acute lymphoblastic leukemia: results from the phase 2/3 AALL1931 study.

AALL1931, a phase 2/3 study conducted in collaboration with the Children's Oncology Group, investigated the efficacy and safety of JZP458 (asparaginase erwinia chrysanthemi [recombinant]-rywn), a recombinant Erwinia asparaginase derived from a novel expression platform, in patients with acute lymphoblastic leukemia/lymphoblastic lymphoma who developed hypersensitivity/silent inactivation to Escherichia coli-derived asparaginases. Each dose of a pegylated E coli-derived asparaginase remaining in patients' treatment plan was substituted by 6 doses of intramuscular (IM) JZP458 on Monday/Wednesday/Friday (MWF). Three regimens were evaluated: cohort 1a, 25 mg/m2 MWF; cohort 1b, 37.5 mg/m2 MWF; and cohort 1c, 25/25/50 mg/m2 MWF. Efficacy was evaluated by the proportion of patients maintaining adequate nadir serum asparaginase activity (NSAA ≥0.1 IU/mL) at 72 hours and at 48 hours during the first treatment course. A total of 167 patients were enrolled: cohort 1a (n = 33), cohort 1b (n = 83), and cohort 1c (n = 51). Mean serum asparaginase activity levels (IU/mL) at 72 hours were cohort 1a, 0.16, cohort 1b, 0.33, and cohort 1c, 0.47, and at 48 hours were 0.45, 0.88, and 0.66, respectively. The proportion of patients achieving NSAA ≥0.1 IU/mL at 72 and 48 hours in cohort 1c was 90% (44/49) and 96% (47/49), respectively. Simulated data from a population pharmacokinetic model matched the observed data well. Grade 3/4 treatment-related adverse events occurred in 86 of 167 (51%) patients; those leading to discontinuation included pancreatitis (6%), allergic reactions (5%), increased alanine aminotransferase (1%), and hyperammonemia (1%). Results demonstrate that IM JZP458 at 25/25/50 mg/m2 MWF is efficacious and has a safety profile consistent with other asparaginases. This trial was registered at www.clinicaltrials.gov as #NCT04145531.