Erwinia Chrysanthemi Research Articles

Beyond the Basics: Taxonomic Classification and Pathogenomics in Recently Discovered Dickeya dadantii Isolates

The genus Dickeya consists of Gram-negative bacteria capable of causing soft rot symptoms in plants, which involves tissue breakdown, particularly in storage organs such as tubers, rhizomes, and bulbs. These bacteria are ranked among the top ten most relevant phytopathogens and seriously threaten economically valuable crops and ornamental plants. This study employs a genomic analysis approach to taxonomically classify and characterize the resistome and virulome of two new strains, CCRMP144 and CCRMP250, identified as Dickeya dadantii. These strains were found to be the causative agents of soft rot symptoms in chili pepper (Capsicum spp.) and lettuce (Lactuca sativa), respectively, in the northeastern region of Brazil. The methodology employed in silico techniques, including tetra correlation search (TCS) and Average Nucleotide Identity (ANI) analysis, in association with a phylogenomic tree inference. TCS and ANI analysis showed that the studied strains belong to the Dickeya dadantii species. The phylogenomic analysis grouped the studied strains in the D. dadantii clade. The genomic characterization demonstrates 68 virulence genes, 54 resistances of biocide and heavy metal genes, and 23 antibiotic resistance genes. As far as we know, this is the first genomic study with Brazilian D. dadantii strains. This study demonstrates the efficacy to taxonomic classification and provides insights into the pathogenesis, host range, and adaptability of these strains which are crucial for the development of more effective management and control strategies for soft rot diseases.

Regulation of bacterial virulence genes by PecS family transcription factors.

Bacterial plant pathogens adjust their gene expression programs in response to environmental signals and host-derived compounds. This ensures that virulence genes or genes encoding proteins, which promote bacterial fitness in a host environment, are expressed only when needed. Such regulation is in the purview of transcription factors, many of which belong to the ubiquitous multiple antibiotic resistance regulator (MarR) protein family. PecS proteins constitute a subset of this large protein family. PecS has likely been distributed by horizontal gene transfer, along with the divergently encoded efflux pump PecM, suggesting its integration into existing gene regulatory networks. Here, we discuss the roles of PecS in the regulation of genes associated with virulence and fitness of bacterial plant pathogens. A comparison of phenotypes and differential gene expression associated with the disruption of pecS shows that functional consequences of PecS integration into existing transcriptional networks are highly variable, resulting in distinct PecS regulons. Although PecS universally binds to the pecS-pecM intergenic region to repress the expression of both genes, binding modes differ. A particularly relaxed sequence preference appears to apply for Dickeya dadantii PecS, perhaps to optimize its integration as a global regulator and regulate genes ancestral to the acquisition of pecS-pecM. Even inducing ligands for PecS are not universally conserved. It appears that PecS function has been optimized to match the unique regulatory needs of individual bacterial species and that its roles must be appreciated in the context of the regulatory networks into which it was recruited.

Pathogenicity of Pectobacterium chrysanthemi on Solanum tuberosumand PCR of the internal transcribed spacer (ITS) 16S/23S rDNA in isolates of Musa sp.from northern Peru

Pectobacterium chrysanthemiis the causative agent of soft rot that commonly affects organic banana crops around the planet. Current phytosanitary control policies are based on the symptoms caused by the disease. However, since there are other species that cause similar symptoms, pathogenicity tests are used to which biochemical tests must be added to identify the bacteria, dispensing with molecular tests that, in contrast, would allow obtaining more specific results and in less time. time. This article presents an alternative methodology for the rapid and specific diagnosis of soft rot caused by Pectobacterium chrysanthemiin organic banana crops in Peru. Detection by virulence of the bacteria in host tissue ofSolanum tuberosumand PCR of the internal transcribed spacer (ITS) of the 16S/23S rDNA allows timely management of the disease. The results demonstrate that in the complex between the pathogenicity test and the ITS-16S/23S PCR assay it is possible to detect Pectobacterium chrysanthemiin a period of 8 days, 3 hours and 15 minutes, demonstrating that this method is more efficient than conventional tests. biochemical tests usually used for the diagnosis of soft rot in Musa sp.

Synergistic enzyme assisted release of ferulic acid from brewer´s spent grain

Brewer’s spent grain (BSG), the most abundant by-product of the beer industry, can be considered a sustainable source of ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA). FA is esterified to the arabinoxylan in the BSG. FA exhibits a variety of biological activities and is a target for valorization of BSG. We present a novel enzyme-assisted approach that utilizes the synergistic action of a specific bacterial endo-1,4-β-xylanase and a fungal ferulic acid esterase activity to selectively release high yields of FA from BSG. The study compares the efficacy of three different fungal feruloyl esterases (E.C. 3.1.1.73) of carbohydrate esterase family 1 (CE1), HiFae, AoFae1, and AnFae1, acting separately or in combination with two different endo-1,4-β-xylanases (EC 3.2.1.8), a bacterial GH30 enzyme from Dickeya chrysanthemi, and Shearzyme®, a commercial GH10 enzyme from Aspergillus aculeatus, respectively. On an equimolar dosage basis, the AnFae1-GH30 was the most efficient combination in releasing FA from BSG. This combination showed significant synergy in a dose-dependent manner, enabling up to 85% FA recovery from BSG within 30 min. of treatment. The results demonstrate a novel enzymatic approach for valorization of BSG as FA is considered a high-value natural compound for use in e.g. anti-aging skin care products.

First Report of Dickeya dadantii Causing Potato Blackleg in Fujian Province China.

Blackleg and soft rot are harmful diseases in potato (Solanum tuberosum) caused by Pectobacterium spp. and Dickeya spp. (Czajkowski et al. 2015). The occurrence of potato blackleg was serious in potato-producing areas around Xiapu County in Fujian Province, China, in 2021 (6 ha) and 2022 (7 ha), with an incidence of approximately 5%, which reached nearly 23%. Three diseased plants were collected to isolate the pathogen. Single colonies from each sampled plant were isolated and streaked onto fresh plates. DNA from three colonies from different plants was PCR amplified with primer pair 27F/1492R (Lane 1991) for the 16S rRNA gene. Since the sequences were identical, we selected strain M2-3 for further analysis. The strain M2-3 was gram-negative, pectolytic on CVP, grew at 37°C and 5% NaCl. The bacterium was positive for phosphatase activity, erythromycin sensitivity, indole production, gelatin liquefaction, malonic utilization, and acid production from, melibiose, raffinose, and arabinose. The bacterium was negative for sucrose, α-methyl glucoside, sorbitol, trehalose, lactose, and sodium citrate (Fujimoto et al. 2018;),although sucrose and lactose did not provide the expected results, there are exception in all species. The genome of strain M2-3 was sequenced and deposited in the NCBI database under accession numbers: CP077422. An Average Nucleotide Identity (ANI) analysis showed that M2-3 clustered with other D. dadantii strains and has a 98.39% identity with D. dadantii strain DSM 18020 (CP023467). The housekeeping genes (recA, dnaX, acnA, gapA, icd, mdh, mtlD and pgi) were amplified with primer pairs designed previously(Fujimoto et al. 2018; Ma et al. 2007) and sequenced. A multilocus sequence analysis (MLSA) was performed by concatenating the 8 gene sequences and constructing a maximum likelihood phylogenetic tree using PhyloSuite version 1.2.1 (Zhang et al. 2020) and IQ-tree version 1.6.8 (Nguyen et al. 2015) software. Strain M2-3 was clustered together with Dickeya dadantii. For the pathogenicity test, three plants per treatment, totaling nine plants, were used. Bacterial suspensions (1×10^8 CFU/mL) were made in a 10mM PBS buffer. 10 μL of M2-3, D. dadantii type strain 18020 (positive control), and buffer (negative control) were injected into the plant stems near the base. Water stains appeared at the site of inoculation after 2 days and they gradually became black and rotten. The leaves became yellow and wilted, and the petiole base rotted within 5 days of inoculation completing the Koch postulate. According to average nucleotide identity and housekeeping gene sequence analysis, strain M2-3 was identified as Dickeya dadantii. Previous studies have reported several pathogens that cause potato blackleg in China, including P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense (Li-ping et al. 2020; Wang et al. 2021). To the best of our knowledge, this study is the first to report potato blackleg disease caused by Dickeya dadantii in Fujian Province, China. This finding suggests that this pathogen may cause a threat to potato production in Fujian Province.

Surviving the Potato Stems: Differences in Genes Required for Fitness by Dickeya dadantii and Dickeya dianthicola.

Bacteria belonging to the genus Dickeya cause blackleg and soft rot symptoms on many plant hosts, including potato. Although there is considerable knowledge about the genetic determinants that allow Dickeya to colonize host plants, as well as the genes that contribute to virulence, much is still unknown. To identify the genes important for fitness in potato stems, we constructed and evaluated randomly barcoded transposon mutant (RB-TnSeq) libraries of Dickeya dadantii and Dickeya dianthicola. We identified 169 and 157 genes important for growth in D. dadantii and D. dianthicola in stems, respectively. This included genes related to metabolic pathways, chemotaxis and motility, transcriptional regulation, transport across membranes, membrane biogenesis, detoxification mechanisms, and virulence-related genes, including a potential virulence cluster srfABC, c-di-GMP modulating genes, and pectin degradation genes. When we compared the results of the stem assay with other datasets, we identified genes important for growth in stems versus tubers and in vitro conditions. Additionally, our data showed differences in fitness determinants for D. dadantii and D. dianthicola. These data provide important insights into the mechanisms used by Dickeya when interacting with and colonizing plants and thus might provide targets for management.

Inoculation Methods to Determine Resistance of Phalaenopsis amabilis (L.) Regeneratded from Irradiated Protocorms to Dickeya dadantii

Soft-rot disease (SRD) in Phalaenopsis, caused Dickeya dadantii, has resulted in significant losses in the orchid sector in Indonesia. This study aimed to evaluate the inoculation method of Dickeya dadantii and identify the resistance response of individual regenerated plantlets of Phal. amabilis from irradiated protocorm. A detached leaf assay was used to evaluate the inoculation method and resistance response of SRD. Based on the results of this study, Dickeya dadantii bacteria could only infect the leaves through wounding tissue. The density of bacteria that could infect leaf tissue was OD600 = 0.2. All dilution factors tested caused soft rot symptoms in P. amabilis. On the other hand, Vanilla planifolia only showed symptoms at a dilution factor of 10-0. Four accessions of regenerated plantlets from irradiated protocorms were resistant to SRD. They were from irradiation 5 Gy (IP 05 Gy-23, IP 05 Gy-31, and IP 05 Gy-33) and one accession from the control treatment or without irradiation (IP 0 Gy -1). These results showed that 5 Gy irradiation increased plant resistance to SRD in Phalaenopsis. A dose of 5 Gy can potentially produce mutant lines resistant to SRD in Phalaenopsis or other plants, too.

Bacteria-antagonists of the agents of soryz bacterial diseases

Aim. To isolate and identify bacteria with antagonist properties for biocontrol of the agents of bacterial diseases of soryz (Sorghum oryzoidum) and sorghum crops. Methods. The studies were conducted in 2021-2023. Spore-forming bacteria were isolated from the soryz samples, collected in the fields of the experimental farm of the Uman National Horticulture University (Cherkasy region, Uman). Lactic acid bacteria were isolated from soryz plants, collected in the private land plot, located between the villages of Teolyn, Vladyslavchyk, Kniazhyky in Monastyryshche com- munity, Uman district, where Pershotravneve hamlet used to be situated. A total of 1,250 samples were analyzed. The experiment had three repeats. Spore-forming and lactic acid bacteria were isolated from the surface of soryz plants on the firm ripe stage in summer while isolating phytopathogenic bacteria. The isolates of lactic acid bacteria- antagonists were also isolated from the inner part of winter stubble stalk of soryz, collected from the tilled field. The antagonistic activity of the strains of lactic acid bacteria and spore-forming bacteria, isolated from different ecological niches, to phytopathogens of soryz and sorghum crops was determined in vitro. The strains of Pseudomonas syringae, the agents of soryz bacterial spots, were used as test-cultures: 211141a, 211141, 210341, 21034, and 210521, along with the collection strains of phytopathogens: Pseudomonas syringae 8299, Pseudomonas syringae subsp. syringae UKM B-1021, X. oryzae 8375, Dickeya chrysanthemi 8683, Diskeya chryzanthemi 8683. The antagonistic activity of the extracted isolates of spore-forming and lactic acid bacteria was studied using the method of radial strokes (joint cultivation of the antagonist and the strains under investigation). The bacterial isolates were deemed inactive if the growth delay zone was 0–5 mm (–), from 5 to 10 mm (+) – low activity, 11–20 mm (++) – moderate activity, over 20 mm (+++) – high activity regarding the test-cultures. To check the effect of the isolate-antagonist of phytopathogenic bacteria, artificial infecting was conducted in the field conditions. For this purpose, a diurnal culture of the antagonist was introduced into the stalk of plants in the concentration of 1×108 colony-forming units, and 24 h later, a culture of test-strain of the phytopathogen was administered above the previous puncture. The results were evaluated 7–14 days after the artificial infection. The experiment had three repeats. The isolates of bacteria which demonstrated their an- tagonistic properties regarding the phytopathogenic bacteria were identified by their morphological properties, Gram staining, catalase test, profile of carbohydrate fermentation and mass-spectrometry (MALDI-TOF – Matrix Assisted Laser Desorption/Ionization) using VITEK MS mass-spectrometer. Results. Thirty-eight spore-forming bacterial iso- lates were extracted from soryz; among these, 21030, 21095, 21040, ASV1, ASV3, B4 demonstrated their antagonistic activity towards the investigated phytopathogenic bacteria. Isolate 21040 showed high antagonistic activity to most test-strains of P. syringae from soryz (the zone of negative culture – 23–30 mm) and lower activity regarding the collection cultures. Isolates B4 and AVS3 demonstrated their selective activity regarding the investigated phytopatho- gens. Twenty isolates of lactic acid bacteria were extracted. Higher antagonistic activity was noted for the isolates of lactic acid bacteria 8/1 and F1 to the strains of P. syringae, isolated from soryz and collection cultures. The highest antagonistic activity of isolate 8/1 was noted regarding test-strains of P. syringae 210521 and X. oryzae 8375 (the zone of negative culture – 40–35 mm). In the field conditions, the treatment of sorghum plants with F1 affected the pathological process that developed due to the impact of the phytopathogenic bacteria P. syringae, which led to the reduction in disease symptoms. The taxonomic position of the isolates of bacteria, which seem to be promising for the control of disease agents, was determined. In terms of morphology of cells and colonies, the biochemical profile, and mass-spectrometry MALDI-TOF, the spore-forming isolates 21040 and B4 were identified as Bacillus subtilis, and ASV3 – as Bacillus vallismortis. The identified isolates of lactic acid bacteria were Lactobacillus pentosus F1 and Lactobacillus sakei 8/1. Conclusions. In addition to phytopathogenic bacteria, from soryz plants we isolated the strains of spore-forming bacteria Bacillus subtilis 21040, B4, Bacillus vallismortis AVS3 and such lactic acid bacteria as Lactiplantibacillus pentosus and Lactobacillus sakei 8/1 (Latilactobacillus sakei 8/1), promising for the elaboration of methods for the biocontrol of the agents of bacterial diseases.

First Report of Dickeya dadantii Causing Soft Rot in Cariru in Brazil.

Cariru (Talinum fruticosum) is a non-conventional food plant and a source of vitamins A, B2, B5, and C, calcium, phosphorus, and iron. It is an important crop for producers of the Amazon region and it is used as a leafy green vegetable, similar to spinach, as well as for medicinal purposes. It is cultivated by producers in the states of Pará and Amazonas (Amorim et al. 2014). In February 2020, in gardens of approximately 1.0 and 0.18 ha in the municipality of Ananindeua (01° 34' 12" S - 48° 38' 11" W), Pará, Brazil, we observed approximately 10% of the plants exhibited soft rot symptoms, bacterial oozing, and stem pith disintegration. From cariru infected leaves, the bacteria were selectively isolated using healthy pepper fruit, as described by Moraes et al. (2017). Two strain (UFRADD17 and UFRADD18) were obtained and both showed white, small, "broken glass" colonies on CPG (peptone-casamino acid-glucose) medium when observed in a stereoscope under oblique lighting. Pathogenicity tests were performed on 20-day-old cariru seedlings by depositing 10 ul of the bacterial suspension (106 CFU/ml) onto a wound made on the leaf blade with an entomological pin. After inoculation, seedlings were incubated in a greenhouse for 48 h at 30 ± 2°C and 90 ± 2% RH. Plants treated similarly with sterile distilled water were used as a negative control. Symptoms of soft rot were observed between 12 and 24 h after inoculation. Seedlings used as negative control remained symptomless. Forty-eight hours after inoculation, we reisolated the pathogen and performed rep-PCR (REP, ERIC, and BOX-PCR) analyses (Gama et al. 2018) with the strains used for inoculation and those reisolated from the pathogenicity test to confirm the identity of the strains and to fulfill Koch's postulates. The two re-isolated strains showed the same REP, ERIC, and BOX-PCR profiles as the strains used for inoculation. In addition, there were no differences between the rep-PCR profiles of the isolates UFRADD17 and UFRADD18. These isolates were Gram-negative, grew at 37°C, and were positive for maceration in pepper fruit and potato tubers. Molecular identification of the isolate UFRADD18 was performed from sequencing fragments from the 16S rDNA region and dnaX, fusA, gyrA, and mdH housekeeping genes as previously described (Van der Wolf et al. 2014; Ma et al. 2007). Sequences were deposited in Genbank under accessions OP142347, OP191704, OP191705, OP191706, and OP191707. Blastn analysis showed 96.15% identity with the 16S rDNA region of accession KY231142.1 (400/416 bp), 99.5% identity with dnaX of accession KC844490.1 (396/398 bp), 99.86% identity with fusA of accession CP023467.1 (714/715 bp), 99.74% identity with gyrA of accession KC844598 (387/388 bp), and 99.10% identity with mdH of accession GQ891979.1 (563/560 bp) from the type strain of Dickeya dadantii. A phylogenetic analysis performed by Bayesian inference with the dnaX, fusA, gyrA, and mdH genes grouped the isolate UFRADD18 along with NCPPB898T, with a 1.00 posterior probability. To our knowledge, this is the first report of D. dadantii causing soft rot in cariru in the Brazilian territory. In addition, this report increases understanding of the host range of this bacterium, which is important for adopting management strategies based on the control of alternative hosts.

Agrobacteria deploy two classes of His-Me finger superfamily nuclease effectors exerting different antibacterial capacities against specific bacterial competitors.

The type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. The Agrobacterium tumefaciens species complex is a group of soil inhabitants and phytopathogens that deploys T6SS as an antibacterial weapon against bacterial competitors at both inter-species and intra-species levels. The A. tumefaciens strain 1D1609 genome encodes one main T6SS gene cluster and four vrgG genes (i.e., vgrGa-d), each encoding a spike protein as an effector carrier. A previous study reported that vgrGa-associated gene 2, named v2a, encodes a His-Me finger nuclease toxin (also named HNH/ENDO VII nuclease), contributing to DNase-mediated antibacterial activity. However, the functions and roles of other putative effectors remain unknown. In this study, we identified vgrGc-associated gene 2 (v2c) that encodes another His-Me finger nuclease but with a distinct Serine Histidine Histidine (SHH) motif that differs from the AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation in Escherichia coli using DNAse activity assay and fluorescence microscopy. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues the phenotypes of growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retains the cell elongation phenotype, while V2a induces cell elongation in a DNase-mediated manner. We also showed that the amino acids of conserved SHH and HNH motifs are responsible for the V2c DNase activity in vivo and in vitro. Notably, V2c also mediated the DNA degradation and cell elongation of the target cell in the context of interbacterial competition. Importantly, V2a and V2c exhibit different capacities against different bacterial species and function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen, Dickeya dadantii.

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

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. 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. 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. 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.

Unravelling the aetiology of Dickeya zeae using polyphasic approaches for bacterial stalk rot in maize

AbstractBacterial stalk rot (BSR), caused by Dickeya zeae (syn. Erwinia chrysanthemi pv. zeae), has emerged as a significant disease affecting maize crops worldwide. In this study, symptomatic maize plants were collected from diverse agroclimatic zones in India over the period of kharif 2019–2021. Various approaches, including pathogenicity tests, cultural characteristics, biochemical profiling and molecular analysis, were employed to accurately identify the collected bacterial isolates as Dickeya. Pathogenicity assessments were conducted on 40‐day‐old maize plants, following Koch's postulates, as well as through potato maceration assays. Furthermore, cross‐infectivity studies were conducted on rice, potato, tomato and banana plants. Phenotypic and biochemical characterization confirmed that all the isolates belonged to the Dickeya genus. Additionally, PCR amplification of a pel gene fragment, specific to the genus Dickeya, further verified the pathogenic isolates as Dickeya. Molecular characterization studies were performed on four isolates (UKMDZ‐3, PBMDZ‐7, TSMDZ‐11 and HPMDZ‐16), selected to represent distinct maize agroclimatic zones and four states of India, and which caused severe infections on susceptible maize cv. Early Composite. Amplification of six characteristic genome regions (16S rRNA, recN, gyrB, dnaX, recA and dnaJ) from these isolates facilitated individual and concatenated gene phylogenetic analyses, confirming their resemblance to Dickeya zeae. This study represents the first comprehensive molecular analysis of D. zeae isolates from India, providing valuable insights for future crop improvement strategies. The findings contribute to our understanding of the genetic basis of BSR in maize and offer potential avenues for genetic enhancement to mitigate the disease's impact on maize cultivation.

Occurrence of Carbapenem-Resistant Enterobacteriaceae (Cre) Abhorring Bla Imp, Bla Kpc And Bla Oxa-48 Genes Recovered From Ready -To- Dispose Poultry Wastes In Osogbo, Osun State

The uncontrolled use of antimicrobials in animal husbandry such as poultry for prophylaxis reasons is to increase output for monetary gains which have led to several acquired community infections with high mortality and morbidity rates. This study is however aimed at investigating the occurrence, prevalence, and molecular detection of carbapenemase genes of carbapenem-resistant Enterobacteriaceae (CRE) bacteria isolated from poultry liters in Osogbo, Osun State Nigeria. Twenty ready-to-pack poultry waste samples were plated for the isolation of Enterobacteriaceae on MacConkey and Brilliance Escherichia coli agar. Disc diffusion method was employed for screening carbapenem resistant isolates while resistant genes were detected using carbapenem resistant gene primers. Forty-six Enterobacteriaeceae were isolated namely: Kluyvera ascorbate (28.3%), Klebsiella oxytoca (10.9%), Citrobacter koseri (8.7%), Klebsiella aereogenes, Raoultell (Klebsiella) planticola, Serratia nematodipila (6.5%), Trabulsiella laguamensis, Salmonella spp. (diarizonae), Proteus vulgaris, Citrobacter farmeri (4.3%), of which Edwardsiella tarda, Providencia rustigianii, Citrobacter freundii, Enterobacter cloacae, Cedecea lapagei, Serratia liquefaciens and Dickeyachry santhemi was (2.2%) respectively. 52.2% of the isolates were carbapenem resistant while 92% of the CRE had bla IMP genes. Only one CRE (Trabulsiella guamensis) was positive to bla KPC gene while four 16.6% of the isolates namely: Citrobacter koseri, Raoultell (Klebsiella) planticola, Serratia nematodipila, Citrobacter freundiu and Citrobacter koseri were positive to blaOXA-48 gene. Therefore, this study infers the high prevalence of CRE isolates in poultry litters as a public health concern for the indiscrimate use of carbapenem.
 Stamford Journal of Microbiology, 2023. Vol. 13, Issue 1, p. 15-20

A comparative study on chemical composition and antibacterial activity of essential oils from the aerial parts and roots of Laportea bulbifera (Siebold & Zucc.) Wedd. growing in the Central Himalayan region

The GC and GC/MS analysis of the essential oil samples obtained separately from whole aerial parts and the roots of Laportea bulbifera resulted in the identification of a total of 40 compounds from the whole aerial parts and 45 compounds from the roots representing 89.7% and 82.9%, respectively, of the total essential oil composition. The essential oil from aerial parts is dominated by oxygenated sesquiterpenes (66.4%) with α-elemol (55.1%), germacrene D-4-ol (4.2%), linalool (3.5%) and phytol (3.4%) as the major constituents while the root essential oil is dominated by oxygenated monoterpenes (30.4%) with trans-myrtanol (16.3%), cis-myrtanol (11.6%), nopinone (7.8%), cadin-4-en-10-ol (4.7%), and α-elemol (3.4%) as major constituents. The essential oil from whole aerial parts showed bactericidal effect at 250 µL/mL concentration (MBC) against Pasteurella multocida and Dickeya dadantii while the root essential oil exhibited bactericidal effect at 125 µL/mL against Escherichia coli, Enterococcus faecalis, Xanthomonas phaseoli, and Dickeya dadantii.

Erwinia amylovora Type III Secretion System Inhibitors Reduce Fire Blight Infection Under Field Conditions.

Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.

Soft rot pathogen Dickeya dadantii 3937 produces tailocins resembling the tails of Peduovirus P2

Tailocins are nanomolecular machines with bactericidal activity. They are produced by bacteria to contribute to fitness in mixed communities, and hence, they play a critical role in their ecology in a variety of habitats. Here, we characterized the new tailocin produced by Dickeya dadantii strain 3937, a well-characterized member of plant pathogenic Soft Rot Pectobacteriaceae (SRP). Tailocins induced in D. dadantii were ca. 166 nm long tubes surrounded by contractive sheaths with baseplates having tail fibers at one end. A 22-kb genomic cluster involved in their synthesis and having high homology to the cluster coding for the tail of the Peduovirus P2 was identified. The D. dadantii tailocins, termed dickeyocins P2D1 (phage P2-like dickeyocin 1), were resistant to inactivation by pH (3.5–12), temperature (4–50°C), and elevated osmolarity (NaCl concentration: 0.01–1 M). P2D1 could kill a variety of different Dickeya spp. but not any strain of Pectobacterium spp. tested and were not toxic to Caenorhabditis elegans.

A Real-World, Single Institution, Post-Market Comparison of Pegaspargase and Calasparagase Pegol-Mknl Therapeutic Drug Monitoring and Toxicity

Introduction: Calaspargase pegol-mknl (CalPEG) was FDA-approved in December 2018 as a component of a multi-agent chemotherapeutic regimen for acute lymphoblastic leukemia/lymphoma (ALL/LLy) in pediatric patients and adolescent and young adults (AYA). It has recently been incorporated into national pediatric ALL consortia clinical trials. The pharmacokinetics and toxicity profiles were reported in two clinical trials (AALL07P4; Angiolillo et al, J Clin Oncol, 2014 and DFCI 11-001; Vrooman et al, J Clin Oncol, 2021), with similar safety profiles to historical pegaspargase (PEGasp). There is a paucity of published data regarding serum asparaginase activity (SAA) level variation or toxicity between CalPEG and PEGasp since FDA-approval. This is timely given the recent transition in asparaginase product availability, which may impact resource utilization, comprehensive drug safety monitoring, and clinical practice guidelines. Methods: We conducted a single institution retrospective review of children and AYA with ALL/LLy who received an asparaginase product during upfront or relapse therapy between January 2022 to June 2023 as part of our chemotherapy quality and safety monitoring (Table). Patients received either PEGasp, CalPEG, and/or asparaginase Erwinia chrysanthemi recombinant (Rylaze) and had correlative SAA documented by a validated assay (NEXT Molecular Analytics; Chester, VA). The Common Terminology Criteria for Adverse Events version 5.0 was used to retrospectively grade asparaginase-related toxicities, including pancreatitis (≥ grade 3), venous thromboembolism (VTE; ≥ grade 2), hyperbilirubinemia (total bilirubin ≥ 5 mg/dL), allergy (≥ grade 3), and silent inactivation (SI, SAA < 0.025 IU/mL within 7 days post-treatment without allergic symptoms). Fisher's exact and Wilcoxon rank-sum tests were used to compare patient characteristics and toxicity incidence with pairwise post-hoc comparisons. P values were two-sided and considered significant if < 0.05. Generalized linear modeling (GLM) was used to model SAA over time and interactions among asparaginase product, toxicity, and immunophenotype. Results: One hundred twenty-five patients received either PEGasp, CalPEG, and/or Rylaze. (Table). Frequency of total asparaginase-related toxicity was comparable between CalPEG (27.7%) and PEGasp (27.4%). However, the incidence of pancreatitis was significantly higher in patients who received CalPEG (12.8%) compared to PEGasp (2.4%), P=0.025, with an Odds Ratio (OR) 5.91. T-ALL/LLy patients who received CalPEG had an increased incidence of both pancreatitis and VTE (33.3% each), with statistically significant pairwise interactions to PEGasp and/or B-ALL cohorts. Conversely, allergy was significantly higher in PEGasp (17.9%) compared to CalPEG (4.3%), P=0.03, with an OR 4.46. SI was found in 5.8% of PEGasp with none yet identified in CalPEG patients. The median peak SAA level over the first 10 days was not different among patients with adverse events; nor were median SAA levels higher in CalPEG patients, despite increased incidence in treatment-related toxicity (Figure). GLM fit slopes were predictably less negative in CalPEG compared to those of PEGasp; patients with toxicity had a larger modeled area under the curve. In 6 patients who were dose-capped to 3750 IU for body mass index (BMI) ≥ 40, all exhibited therapeutic SAA without any asparaginase-related toxicity. Conclusions: To our knowledge this is the first single-institution post-market retrospective quality and safety analysis comparing CalPEG and PEGasp therapeutic drug monitoring and toxicity. Interestingly, the incidence of pancreatitis and VTE is significantly higher in patients receiving CalPEG and disproportionately higher in T-lineage. Median peak SAA levels were not predictive of toxicity, though GLM may help provide a predictive model. The rate of SI emphasizes the need for continued resource utilization of SAA monitoring. Our observed differences in CalPEG and PEGasp toxicity are discordant with two previously published clinical trials used for FDA approval of CALPEG (AALL07P4 and DFCI 11-001). Larger analyses of CalPEG SAA and toxicities are necessary to determine if these trends are reproducible. This information will help establish optimal utilization of SAA safety monitoring with the broader goal of identifying necessary practice changes in CalPEG clinical management.

Development of a bioengineered Erwinia chrysanthemi asparaginase to enhance its anti-solid tumor potential for treating gastric cancer

Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for ∼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.

Antibacterial Effect of Juglans regia, Citrus sinensis, Vicia faba, and Urtica urens Extracts under In vitro Conditions

Various agricultural products are known to have anti-microbial, anti-cancer, and anti-inflammatory effects. As we can mention, Walnut (Juglans regia) husk, orange (Citrus sinensis) peel, Broad bean (Vicia faba) peel, and Nettle (Urtica urens) are proven to have antimicrobial and anticancer actions. Also, plant diseases such a Pectobacterium carotovorum, Ralstonia solanacearum, Dickeya chrysanthemi, and Pseudomonoas syringae are known to cause annual damage to plant products. Therefore, in the current study, the researchers evaluated their antibacterial activity on the mentioned bacteria under in vitro conditions. Extracts of J. regia husk, C. sinensis peel, V. faba outer peel and U. urens were prepared by maceration method and their anti-bacterial activity on P. carotovorum, D. chrysanthemi, P. syringae, R. solancearum was evaluated using blank disk and well diffusion to obtain minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. J. regia (husk) and V. faba (outer peel) extracts revealed an anti-bacterial effect on all 4 studied bacteria, while C. sinensis (peel) only inhibited the growth of P. syringae and U. urens only had this effect on P. syringae and R. Solancearum. The inhibition zones varied from 8 to 14 mm, while almost all MIC and MBC rates were 6.25 and 12.5 mg/mL, respectively. Based on the antimicrobial results, the extracts that showed suitable antibacterial effects on certain bacteria can be further studied to be used as natural pesticides.

Dissecting the molecular dance: c-di-GMP, cAMP-CRP, and VfmH collaboration in pectate lyase regulation for Dickeya dadantii-unveiling the soft rot pathogen's strategy.

Dickeya dadantii is a Gram-negative bacterium that causes soft rot diseases in a variety of plants. This pathogen secretes pectate lyase (Pel) enzymes to degrade the plant cell wall, a process controlled by the bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP). VfmH is a Fis-family response regulator of the Vfm quorum sensing system, which has been previously reported to positively regulate pel transcription. Our results demonstrated that VfmH upregulated the production of Pel at low c-di-GMP levels, which was suppressed by c-di-GMP via deleting gene egcpB, encoding a phosphodiesterase that degrades c-di-GMP. Multiple sequence alignments between FleQ, a known c-di-GMP binding protein, and VfmH revealed several potential c-di-GMP binding motifs. We further validated these motifs via single amino acid substitutions. Indeed, derivatives of VfmH, including VfmHR195A, VfmHR279A, and VfmHR344A, abolished the c-di-GMP binding ability and the corresponding regulation in Pel. We found that VfmH exhibited ATPase activity, and this could be hindered by c-di-GMP binding. Interestingly, our results showed that VfmH interacted with CRP, suggesting that the cyclic AMP (cAMP) signaling pathway is also involved in the VfmH-mediated Pel regulation. Taken together, our study suggested that D. dadantii amalgamates both c-di-GMP and cAMP signaling networks along with the Vfm quorum sensing pathway to control Pel.IMPORTANCEBacteria respond to environmental changes and adapt to host systems. The response regulator VfmH of the Vfm quorum sensing system regulates a crucial virulence factor, pectate lyase (Pel), in Dickeya dadantii. At high c-di-GMP concentrations, VfmH binds c-di-GMP, resulting in the loss of its activation property in the Pel and virulence regulation in D. dadantii. VfmH binds to c-di-GMP via three conserved arginine residues, and mutations of these residues eliminate the c-di-GMP-related phenotypes of VfmH in Pel synthesis. Our data also show that VfmH interacts with CRP to regulate pelD transcription, thus integrating cyclic AMP and c-di-GMP signaling pathways to control virulence in D. dadantii. We propose that VfmH is an important intermediate factor incorporating quorum sensing and nucleotide signaling pathways for the collective regulation of D. dadantii pathogenesis.