Whole Genome Sequencing Analysis Research Articles

Molecular basis of the persistence of chloramphenicol resistance among Escherichia coli and Salmonella spp. from pigs, pork and humans in Thailand.

This study aimed to investigate the potential mechanisms associated with the persistence of chloramphenicol (CHP) resistance in Escherichia coli and Salmonella enterica isolated from pigs, pork, and humans in Thailand. The CHP-resistant E. coli (n = 106) and Salmonella (n = 57) isolates were tested for their CHP susceptibility in the presence and absence of phenylalanine arginine β-naphthylamide (PAβN). The potential co-selection of CHP resistance was investigated through conjugation experiments. Whole genome sequencing (WGS) was performed to analyze the E. coli (E329, E333, and E290) and Salmonella (SA448, SA461, and SA515) isolates with high CHP MIC (32-256 μg/mL) and predominant plasmid replicon types. The presence of PAβN significantly reduced the CHP MICs (≥4-fold) in most E. coli (67.9%) and Salmonella (64.9%). Ampicillin, tetracycline, and streptomycin co-selected for CHP-resistant Salmonella and E. coli-transconjugants carrying cmlA. IncF plasmids were mostly detected in cmlA carrying Salmonella (IncFIIAs) and E. coli (IncFIB and IncF) transconjugants. The WGS analysis revealed that class1 integrons with cmlA1 gene cassette flanked by IS26 and TnAs1 were located on IncX1 plasmid, IncFIA(HI1)/HI1B plasmids and IncFII/FIB plasmids. IncFIA(HI1)/HI1B/Q1in SA448 contained catA flanked by IS1B and TnAs3. In conclusion, cross resistance through proton motive force-dependent mechanisms and co-selection by other antimicrobial agents involved the persistence of CHP-resistance in E. coli in this collection. Dissemination of CHP-resistance genes was potentially facilitated by mobilization via mobile genetic elements.

Integrative analysis of genetics, epigenetics and RNA expression data reveal three susceptibility loci for smoking behavior in Chinese Han population.

Despite numerous studies demonstrate that genetics and epigenetics factors play important roles on smoking behavior, our understanding of their functional relevance and coordinated regulation remains largely unknown. Here we present a multiomics study on smoking behavior for Chinese smoker population with the goal of not only identifying smoking-associated functional variants but also deciphering the pathogenesis and mechanism underlying smoking behavior in this under-studied ethnic population. After whole-genome sequencing analysis of 1329 Chinese Han male samples in discovery phase and OpenArray analysis of 3744 samples in replication phase, we discovered that three novel variants located near FOXP1 (rs7635815), and between DGCR6 and PRODH (rs796774020), and in ARVCF (rs148582811) were significantly associated with smoking behavior. Subsequently cis-mQTL and cis-eQTL analysis indicated that these variants correlated significantly with the differential methylation regions (DMRs) or differential expressed genes (DEGs) located in the regions where these variants present. Finally, our in silico multiomics analysis revealed several hub genes, like DRD2, PTPRD, FOXP1, COMT, CTNNAP2, to be synergistic regulated each other in the etiology of smoking.

Molecular epidemiology of invasive Group A streptococcal infections before and after the COVID-19 pandemic in Switzerland

Group A Streptococcus (GAS, aka Streptococcus pyogenes) poses a significant public health concern, causing a diverse spectrum of infections with high mortality rates. Following the COVID-19 pandemic, a resurgence of invasive GAS (iGAS) infections has been documented, necessitating efficient outbreak detection methods. Whole genome sequencing (WGS) serves as the gold standard for GAS molecular typing, albeit constrained by time and costs. This study aimed to characterize the post-pandemic increased prevalence of iGAS on the molecular epidemiological level in order to assess whether new, more virulent variants have emerged. A total of 66 iGAS strains isolated from nine Swiss hospitals during the COVID-19 post-pandemic increased GAS prevalence were evaluated and compared to 15 strains collected before and 12 during the COVID-19 pandemic. FT-IR measurements and WGS were conducted for network analysis. Demographic, clinical, and epidemiological data were collected. Skin and soft tissue infection was the most common diagnosis, followed by primary bacteremia and pneumonia. Viral co-infections were found in 25% of cases and were significantly associated with more severe disease requiring intensive care unit admission. WGS analysis did not reveal emerging GAS genetic distinct variants after the COVID-19 pandemic, indicating the absence of a pandemic-induced shift. FT-IR spectroscopy exhibited limitations in differentiating genetically distant GAS strains, yielding poor overlap with WGS-derived clusters. The emm1/ST28 genotype was predominant in our cohort and was associated with five of the seven deaths recorded, in accordance with the molecular epidemiological data before the pandemic. Additionally, no notable shift in antibiotic susceptibility patterns was observed. Our data suggest that mainly non-pathogen related factors contributed to the recent increased prevalence of iGAS.

Whole-genome sequencing of marine water-derived Curvularia verruculosa KHW-7: a pioneering study

Marine microorganisms are renowned for being a rich source of new secondary metabolites that are significant to humans. The fungi strain KHW-7 was isolated from the seawater collected from the Gulf of Khambhat, India, and identified as Curvularia verruculosa KHW-7. On a next-generation sequencing platform, C. verruculosa KHW-7’s whole-genome sequencing (WGS) and gene annotation were carried out using several bioinformatic methods. The 31.59 MB genome size, 52.3% GC, and 158 bp mean read length were discovered using WGS. This genome also contained 9,745 protein-coding genes, including 852 secreted proteins and 2048 transmembrane proteins. The antiSMASH algorithm used to analyze genomes found 25 secondary metabolite biosynthetic gene clusters (BGCs) that are abundant in terpene, non-ribosomal peptide synthetase (NRPS), and polyketides type 1 (T1PKS). To our knowledge, this is the first whole-genome sequence report of C. verruculosa. The WGS analysis of C. verruculosa KHW-7 indicated that this marine-derived fungus could be an efficient generator of bioactive secondary metabolites and an important industrial enzyme, both of which demand further investigation and development.

Resolving unsolved whole-genome sequencing data in paediatric neurological disorders: a cohort study

ObjectiveTo resolve unsolved whole-genome sequencing (WGS) data in individuals with paediatric neurological disorders.DesignA cohort study method using updated bioinformatic tools, new analysis targets, clinical information and literature databases was employed...

Emergence of novel hypervirulent Acinetobacter baumannii strain and herpes simplex type 1 virus in a case of community-acquired pneumonia in China

BackgroundA. baumannii is an important and common clinical pathogen, especially in the intensive care unit (ICU). This study aimed to characterize one hypervirulent A. baumannii strain in a patient with community-acquired pneumonia and herpes simplex type 1 virus infection. MethodsMinimum inhibitory concentrations (MICs) were determined using the Kirby-Bauer (K-B) and broth microdilution methods. Galleria mellonella infection model experiment was conducted. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The resistance and virulence determinants were identified using the ABRicate program with ResFinder and the VFDB database. The capsular polysaccharide locus (K locus) and lipooligosaccharide outer core locus (OC locus) were identified using Kleborate with Kaptive. Phylogenetic analyses were conducted using the BacWGSTdb server. ResultsA. baumannii XH2146 strain belongs to ST10Pas and ST447Oxf. The strain was resistant to cefazolin, ciprofloxacin, and trimethoprim/sulfamethoxazole (TMP-SMX). Bautype and Kaptive analyses showed that XH2146 contains OCL2 and KL49. WGS analysis revealed that the strain harbored blaADC-76, blaOXA-68, ant(3′’)-IIa, tet(B), and sul2. Notably, tet(B) and sul2, both were located within a 114,700-bp plasmid (designated pXH2146–1). Virulence assay revealed A. baumannii XH2146 possessed higher virulence than A. baumannii AB5075 at 12 h. Comparative genomic analysis showed that A. baumannii ST447 strains were mainly isolated from the USA and exhibited a relatively close genetic relationship. Importantly, 11 strains were observed to carry blaOXA-58; blaOXA-23 was identified in 11 isolates and three ST447 A. baumannii strains harbored blaNDM-1. ConclusionsEarly detection of community-acquired hypervirulent Acinetobacter baumannii strains is recommended to prevent their extensive spread in hospitals.

Genetic characterization of plasmid-borne blaOXA-58 and blaOXA-72 in Acinetobacter pittii in Shaanxi, China

ObjectivesAcinetobacter pittii has emerged as an opportunistic nosocomial pathogen associated with hospital-acquired infections. The purpose of this study was to investigate the genetic structures of plasmids carrying carbapenemase genes blaOXA-58 and blaOXA-72 in A. pittii strains AR3676 and AR3651 isolated from patients. MethodsAntimicrobial susceptibility testing was performed using broth microdilution. Whole-genome sequencing and bioinformatics analysis were performed to characterize the genome of A. pittii AR3676 and AR3651. Conjugation experiments were conducted to evaluate plasmid transferability. Phylogenetic and comparative genomic analysis were performed to explore the characteristics of carbapenem-resistant A. pittii isolates worldwide. ResultsThe AR3676 strain showed resistance to imipenem. The 19 700-bp plasmid pAR3676-OXA-58 harboured blaOXA-58 with genetic contexts consisting of a truncated ISAba3-like-blaOXA58-ISAba3. Additionally, the AR3651 strain showed resistance to imipenem and meropenem. The AR3651 genome comprised one 9,837-bp RepA_AB plasmid pAR3651-OXA-72 harbouring blaOXA-72. This blaOXA-72 was flanked by XerC/XerD recombination sites. The conjugation of plasmids pAR3676-OXA-58 and pAR3651-OXA-72 from A. pittii to Acinetobacter baumannii ATCC 17978RIFR failed three independent times. Phylogenetic analysis of A. pittii strains AR3676, AR3651, and a further 504 A. pittii strains collected between 1966 and 2022 from various geographic localities revealed genetic diversity with a heterogeneous distribution of carbapenemase genes. ConclusionsA. pittii strains with a plasmid carrying blaOXA-58 or blaOXA-72 may serve as an important reservoir of carbapenemase genes. Carbapenemase genes on a single plasmid may facilitate their dissemination and persistence. Additionally, pdif sites and mobile elements play an important role in the mobilization of resistance genes and plasmid evolution.

Facial Skin Microbiome Composition and Functional Shift with Aging.

The change in the skin microbiome as individuals age is only partially known. To provide a better understanding of the impact of aging, whole-genome sequencing analysis was performed on facial skin swabs of 100 healthy female Caucasian volunteers grouped by age and wrinkle grade. Volunteers' metadata were collected through questionnaires and non-invasive biophysical measurements. A simple model and a biological statistical model were used to show the difference in skin microbiota composition between the two age groups. Taxonomic and non-metric multidimensional scaling analysis showed that the skin microbiome was more diverse in the older group (≥55 yo). There was also a significant decrease in Actinobacteria, namely in Cutibacterium acnes, and an increase in Corynebacterium kroppenstedtii. Some Streptococcus and Staphylococcus species belonging to the Firmicutes phylum and species belonging to the Proteobacteria phylum increased. In the 18-35 yo younger group, the microbiome was characterized by a significantly higher proportion of Cutibacterium acnes and Lactobacillus, most strikingly, Lactobacillus crispatus. The functional analysis using GO terms revealed that the young group has a higher significant expression of genes involved in biological and metabolic processes and in innate skin microbiome protection. The better comprehension of age-related impacts observed will later support the investigation of skin microbiome implications in antiaging protection.

Occurrence and characteristics of blaOXA-181-carrying Klebsiella aerogenes from swine in China

Klebsiella aerogenes is a largely understudied opportunistic pathogen that can cause sepsis and lead to high mortality rates. In this study, we reported the occurrence of carbapenem-resistant blaOXA-181-carrying Klebsiella aerogenes from swine in China and elucidate their genomic characteristics. A total of 126 samples, including 109 swine fecal swabs, 14 environmental samples, and three feed samples were collected from a pig farm in China. The samples were enriched with LB broth culture and then inoculated into MacConkey agar plates for bacterial isolation. After PCR detection of carbapenemases genes, the blaOXA-181-carrying isolates were subjected to antimicrobial susceptibility testing, and whole-genome sequence analysis. Four Klebsiella aerogenes isolates carrying the blaOXA-181 gene were obtained from swine faecal samples. All the 4 strains were belonged to ST438. The blaOXA-181 genes were located in IncX3-ColKP3 hybrid plasmids with the core genetic structure of IS26-ΔIS3000-ΔISEcp1-blaOXA-181-ΔlysR-ΔereA-ΔrepA-ISKpn19-tinR-qnrS1-ΔIS2-IS26, which suggests the potential for horizontal transfer and further dissemination of this resistance gene among Enterobacteriaceae and other sources. This study represents the first instance of OXA-181-producing K. aerogenes being identified from swine faeces in China. It is crucial to maintain continuous monitoring and ongoing attention to the detection of K. aerogenes carrying blaOXA-181 and other resistance genes in pigs.

Whole Genome Sequence Analysis of Listeria monocytogenes Isolates Obtained from the Beef Production Chain in Gauteng Province, South Africa.

The study used whole-genome sequencing (WGS) and bioinformatics analysis for the genomic characterization of 60 isolates of Listeria monocytogenes obtained from the beef production chain (cattle farms, abattoirs, and retail outlets) in Gauteng province, South Africa. The sequence types (STs), clonal complexes (CCs), and the lineages of the isolates were determined using in silico multilocus sequence typing (MLST). We used BLAST-based analyses to identify virulence and antimicrobial genes, plasmids, proviruses/prophages, and the CRISPR-Cas system. The study investigated any association of the detected genes to the origin in the beef production chain of the L. monocytogenes isolates. Overall, in 60 isolates of Listeria monocytogenes, there were seven STs, six CCs, forty-four putative virulence factors, two resistance genes, one plasmid with AMR genes, and three with conjugative genes, one CRISPR gene, and all 60 isolates were positive for proviruses/prophages. Among the seven STs detected, ST204 (46.7%) and ST2 (21.7%) were the most prominent, with ST frequency varying significantly (p < 0.001). The predominant CC detected were CC2 (21.7%) and CC204 (46.7%) in lineages I and II, respectively. Of the 44 virulence factors detected, 26 (across Listeria Pathogenicity Islands, LIPIs) were present in all the isolates. The difference in the detection frequency varied significantly (p < 0.001). The two AMR genes (fosX and vga(G)) detected were present in all 60 (100%) isolates of L. monocytogenes. The only plasmid, NF033156, was present in three (5%) isolates. A CRISPR-Cas system was detected in six (10%), and all the isolates carried proviruses/prophages. The source and sample type significantly affected the frequencies of STs and virulence factors in the isolates of L. monocytogenes. The presence of fosX and vga(G) genes in all L. monocytogenes isolates obtained from the three industries of the beef production chain can potentially cause therapeutic implications. Our study, which characterized L. monocytogenes recovered from the three levels in the beef production chain, is the first time genomics was performed on this type of data set in the country, and this provides insights into the health implications of Listeria.

Clinical Course, Antifungal Susceptibility, and Genomic Sequencing of Trichophyton indotineae

Trichophyton indotineae is an emerging dermatophyte causing outbreaks of extensive tinea infections often unresponsive to terbinafine. This species has been detected worldwide and in multiple US states, yet detailed US data on infections with T indotineae are sparse and could improve treatment practices and medical understanding of transmission. To correlate clinical features of T indotineae infections with in vitro antifungal susceptibility testing results, squalene epoxidase gene sequence variations, and isolate relatedness using whole-genome sequencing. This retrospective cohort study of patients with T indotineae infections in New York City spanned May 2022 to May 2023. Patients with confirmed T indotineae infections were recruited from 6 New York City medical centers. Improvement or resolution at the last follow-up assessment. Among 11 patients with T indotineae (6 male and 5 female patients; median [range] age, 39 [10-65] years), 2 were pregnant; 1 had lymphoma; and the remainder were immunocompetent. Nine patients reported previous travel to Bangladesh. All had widespread lesions with variable scale and inflammation, topical antifungal monotherapy failure, and diagnostic delays (range, 3-42 months). Terbinafine treatment failed in 7 patients at standard doses (250 mg daily) for prolonged duration; these patients also had isolates with amino acid substitutions at positions 393 (L393S) or 397 (F397L) in squalene epoxidase that correlated with elevated terbinafine minimum inhibitory concentrations of 0.5 μg/mL or higher. Patients who were treated with fluconazole and griseofulvin improved in 2 of 4 and 2 of 5 instances, respectively, without correlation between outcomes and antifungal minimum inhibitory concentrations. Furthermore, 5 of 7 patients treated with itraconazole cleared or had improvement at the last follow-up, and 2 of 7 were lost to follow-up or stopped treatment. Based on whole-genome sequencing analysis, US isolates formed a cluster distinct from Indian isolates. The results of this case series suggest that disease severity, diagnostic delays, and lack of response to typically used doses and durations of antifungals for tinea were common in this primarily immunocompetent patient cohort with T indotineae, consistent with published data. Itraconazole was generally effective, and the acquisition of infection was likely in Bangladesh.

Marine Pediococcus pentosaceus E3 Probiotic Properties, Whole-Genome Sequence Analysis, and Safety Assessment.

Probiotics play a significant role in enhancing health, and they are well known for bacteriocins production. Evaluating probiotics' whole-genome sequence provides insights into their consumption outcomes. Thus, genomic studies have a significant role in assessing the safety of probiotics more in-depth and offer valuable information regarding probiotics' functional diversity, metabolic pathways, and health-promoting mechanisms. Marine Pediococcus pentosaceus E3, isolated from shrimp gut, exhibited beneficial properties, indicating its potential as a probiotic candidate. Phenotypically, E3 strain was susceptible to most antibiotics assessed, tolerant to low pH and high bile salt conditions, and revealed no hemolysin activity. Interestingly, E3-neutralized CFS revealed significant antibacterial activity against pathogens under investigation. Therefore, the concentrated CFS was prepared and evaluated as a natural biopreservative and showed outstanding antimicrobial activity. Furthermore, integrated-based genome assessment has provided insight into probiotic characteristics at the genomic level. Whole-genome sequencing analysis revealed that the E3 genome possesses 1805 protein-coding genes, and the genome size was about 1.8Mb with a G + C content of 37.28%. Moreover, the genome revealed the absence of virulence factors and clinically related antibiotic genes. Moreover, several genes consistent with probiotic microorganisms' features were estimated in the genome, including stress response, carbohydrate metabolism, and vitamin biosynthesis. In addition, several genes associated with survival and colonization within the gastrointestinal tract were also detected across the E3 genome. Therefore, the findings suggest that insights into the genetic characteristics of E3 guarantee the safety of the strain and facilitate future development of E3 isolate as a health-promoting probiotic and source of biopreservative.

Listeria monocytogenes from Food Products and Food Associated Environments: Antimicrobial Resistance, Genetic Clustering and Biofilm Insights.

Listeria monocytogenes, a foodborne pathogen, exhibits high adaptability to adverse environmental conditions and is common in the food industry, especially in ready-to-eat foods. L. monocytogenes strains pose food safety challenges due to their ability to form biofilms, increased resistance to disinfectants, and long-term persistence in the environment. The aim of this study was to evaluate the presence and genetic diversity of L. monocytogenes in food and related environmental products collected from 2014 to 2022 and assess antibiotic susceptibility and biofilm formation abilities. L. monocytogenes was identified in 13 out of the 227 (6%) of samples, 7 from food products (meat preparation, cheeses, and raw milk) and 6 from food-processing environments (slaughterhouse-floor and catering establishments). All isolates exhibited high biofilm-forming capacity and antibiotic susceptibility testing showed resistance to several classes of antibiotics, especially trimethoprim-sulfamethoxazole and erythromycin. Genotyping and core-genome clustering identified eight sequence types and a cluster of three very closely related ST3 isolates (all from food), suggesting a common contamination source. Whole-genome sequencing (WGS) analysis revealed resistance genes conferring resistance to fosfomycin (fosX), lincosamides (lin), fluoroquinolones (norB), and tetracycline (tetM). In addition, the qacJ gene was also detected, conferring resistance to disinfecting agents and antiseptics. Virulence gene profiling revealed the presence of 92 associated genes associated with pathogenicity, adherence, and persistence. These findings underscore the presence of L. monocytogenes strains in food products and food-associated environments, demonstrating a high virulence of these strains associated with resistance genes to antibiotics, but also to disinfectants and antiseptics. Moreover, they emphasize the need for continuous surveillance, effective risk assessment, and rigorous control measures to minimize the public health risks associated to severe infections, particularly listeriosis outbreaks. A better understanding of the complex dynamics of pathogens in food products and their associated environments can help improve overall food safety and develop more effective strategies to prevent severe health consequences and economic losses.

Unveiling antibiofilm potential: proteins from Priestia sp. targeting Staphylococcus aureus biofilm formation.

Staphylococcus aureus is the etiologic agent of many nosocomial infections, and its biofilm is frequently isolated from medical devices. Moreover, the dissemination of multidrug-resistant (MDR) strains from this pathogen, such as methicillin-resistant S. aureus (MRSA) strains, is a worldwide public health issue. The inhibition of biofilm formation can be used as a strategy to weaken bacterial resistance. Taking that into account, we analysed the ability of marine sponge-associated bacteria to produce antibiofilm molecules, and we found that marine Priestia sp., isolated from marine sponge Scopalina sp. collected on the Brazilian coast, secretes proteins that impair biofilm development from S. aureus. Partially purified proteins (PPP) secreted after 24 hours of bacterial growth promoted a 92% biofilm mass reduction and 4.0 µg/dL was the minimum concentration to significantly inhibit biofilm formation. This reduction was visually confirmed by light microscopy and Scanning Electron Microscopy (SEM). Furthermore, biochemical assays showed that the antibiofilm activity of PPP was reduced by ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline (PHEN), while it was stimulated by zinc ions, suggesting an active metallopeptidase in PPP. This result agrees with mass spectrometry (MS) identification, which indicated the presence of a metallopeptidase from the M28 family. Additionally, whole-genome sequencing analysis of Priestia sp. shows that gene ywad, a metallopeptidase-encoding gene, was present. Therefore, the results presented herein indicate that PPP secreted by the marine Priestia sp. can be explored as a potential antibiofilm agent and help to treat chronic infections.

The influence of an innovative antibacterial drug of the thiadiazinone class on the virulence factors of bacteria of the phylum Pseudomonadota, which chronically infect patients with cystic fibrosis

Introduction. Infections of the lower respiratory tract by bacteria of the Pseudomonadota phylum: Pseudomonas aeruginosa, Burkholderia spp., Achromobacter spp. are critical to the quality and life expectancy of patients with cystic fibrosis (CF). When the infection is chronic, eradication of bacteria with existing antibacterial drugs is practically impossible. To explore alternative drugs, trials are needed on bacteria isolated from CF patients and characterized using genomic approaches. The objective of our study was a comparative analysis of virulence factors of 6 isolates of bacteria of the Pseudomonadota phylum and testing the efficacy of the innovative drug Fluorothiazinone (FT) in suppressing the pathogenicity of bacteria in vitro. Materials and methods. Isolates of A. ruhlandii ST36, A. xylosoxidans ST555, B. cepacia ST2140, B. gladioli ST2141, P. aeruginosa ST859 and ST198 were examined using whole-genome sequencing and bioinformatics analysis to search for resistance and virulence determinants. The FT drug was tested for its effect on bacteria in in vitro experiments on cytotoxicity on HeLa cells, motility and biofilm formation. Results. Genomic studies have confirmed the arsenal of resistance determinants, especially the efflux systems of bacteria isolated from patients with CF, and the diversity of virulence factors, among which we identified factors in the categories of motility, signals of quorum-sensing systems, secretion systems, exotoxins, as the most essential for the adaptation of bacteria to conditions of the lower respiratory tract. In vitro tests of the FT drug showed its effectiveness in suppressing cytotoxicity (2.6–4.0 times), motility (2.0–3.6 times) and the process of biofilm formation (2.0–7.7 times). Conclusion. For the first time, the effectiveness of the innovative antibacterial drug Fluorothiazinone has been shown against bacteria of the Pseudomonadota phylum, isolated from chronically infected patients with CF, with the described potential of virulence factors.

Mechanisms governing melon fruit skin pigmentation: Insights from transcriptome sequencing and whole-genome bisulfite sequencing analyses

Fruit skin color is a critical agronomic trait determining fruit quality, but the mechanisms governing melon (Cucumis melo L.) fruit skin pigmentation remain inadequately characterized. We performed cytological, transcriptomic, and whole-genome bisulfite sequencing (WGBS) comparative analyses on two near-isogenic lines (NILs) at different developmental stages: the green-skinned (GS) and white-skinned (WS) melon. The enriched functions among the 536 identified differentially expressed genes (DEGs) were related to chloroplast development, pigment metabolism, and photosynthetic pathway. The genes in these pathways were downregulated in the WS line, potentially contributing to the decreased chlorophyll content and abnormal chloroplast development in WS. WGBS analysis revealed that the levels of genomic DNA methylation in WS progressively exceeded those in GS as the fruits developed. The integrated analysis of the transcriptome and methylome identified DMR-associated DEGs (CHLI, CRD1, PORA, and HCAR) involved in chlorophyll metabolism. Notably, PORA exhibited downregulated expression and was hypermethylated in WS fruits during two developmental stages, showing a coordinated expression with the chlorophyll contents in developing WS and GS fruits. Furthermore, we identified a transcription factor gene, MELO3C011576, that was closely associated with the expression of PORA and chlorophyll levels across three developmental stages in both NILs. We speculate that PORA positively regulates chlorophyll biosynthesis in the two NILs, with its expression may be co-controlled by DNA methylation and transcription factors.

Identification and Safety Assessment of Enterococcus casseliflavus KB1733 Isolated from Traditional Japanese Pickle Based on Whole-Genome Sequencing Analysis and Preclinical Toxicity Studies.

The present study involves the precise identification and safety evaluation of Enterococcus casseliflavus KB1733, previously identified using 16S rRNA analysis, through whole-genome sequencing, phenotypic analysis, and preclinical toxicity studies. Analyses based on the genome sequencing data confirm the identity of KB1733 as E. casseliflavus and show that the genes related to vancomycin resistance are only present on the chromosome, while no virulence factor genes are present on the chromosome or plasmid. Phenotypic analyses of antibiotic resistance and hemolytic activity also indicated no safety concerns. A bacterial reverse mutation test showed there was no increase in revertant colonies of heat-killed KB1733. An acute toxicity test employing heat-killed KB1733 at a dose of 2000 mg/kg body weight in rats resulted in no deaths and no weight gain or other abnormalities in the general condition of the animals, with renal depression foci and renal cysts only occurring at the same frequency as in the control. Taking the background data into consideration, the effects on the kidneys observed in the current study were not caused by KB1733. Our findings suggest that KB1733 is non-pathogenic to humans/animals, although further studies involving repeated oral toxicity tests and/or clinical tests are required.

Novel oceanic cyanobacterium isolated from Bangaram island with profound acid neutralizing ability is proposed as Leptolyngbya iicbica sp. nov. strain LK

An acid-neutralizing, filamentous, non-heterocytous, marine cyanobacterium named 'LK' has been isolated from the seashore of Bangaram Island, an atoll of Lakshadweep, India, and is described here as a novel species. LK has been characterized using morphological, ecological, and genomic features. Based on 16S rRNA, whole-genome sequencing, and marker gene-based analysis, LK has been identified as a new species. LK clustered with Leptolyngbya-like strains belonging to the LPP group but diverged from Leptolyngbya sensu stricto, indicating the polyphyletic nature of the Leptolyngbya genus. Leptolyngbya sp. SIOISBB and Halomicronema sp. CCY15110 were identified as LK's two closest phylogenetic neighbors in various phylogenetic studies. The analysis of 16S rRNA, ITS secondary structures, and genome relatedness indices such as AAI, ANI, and gANI strongly support LK as a novel species of the Leptolyngbya genus. The mechanism behind acid neutralization in LK has been delineated, attributing it to a surface phenomenon most likely due to the presence of salts of calcium, magnesium, sodium, and potassium. We name LK as Leptolyngbya iicbica strain LK which is a novel species with prominent acidic pH-neutralizing properties.

In vitro, in planta, and comparative genomic analyses of Pseudomonas syringae pv. syringae strains of pepper (Capsicum annuum var. annuum).

Pseudomonas leaf spot (PLS) caused by Pseudomonas syringae pv. syringae (Pss) causes significant losses to the pepper industry. Highly virulent Pss strains under optimal environmental conditions (cool-moderate temperatures, high moisture) can cause severe necrotic lesions on pepper leaves that consequently can decrease pepper yield if the disease persists. Hence, it is important to understand the virulence mechanisms of Pss to be able to effectively control PLS in peppers. In our study, in vitro, in planta, and whole-genome sequence analyses were conducted to better understand the virulence and pathogenicity characteristics of Pss strains in peppers. Our findings fill a knowledge gap regarding potential virulence and pathogenicity characteristics of Pss in peppers, including virulence and antimicrobial gene content. Our study helps pave a path to further identify the role of specific virulence genes in causing disease in peppers, which can have implications in developing strategies to effectively control PLS in peppers.

XA21-mediated resistance to Xanthomonas oryzae pv. oryzae is dose dependent.

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21. These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.