Complete Genome Sequence Analysis Research Articles

The S protein of a novel recombinant PEDV strain promotes the infectivity and pathogenicity of PEDV in mid-west China.

Porcine epidemic diarrhoea virus (PEDV) is an emerging and re-emerging swine enterovirus that causes highly contagious diarrhoea and mortality in piglets. To better understand the current prevalence of PEDV in mid-west China, and to find out the reason for the re-emergence of PEDV from the viral genomic characteristics. Herein, we firstly investigated epidemiology of PEDV in mid-west China from 2019 to 2020. A total of 62.23% (257/413) of diarrhoea samples were positive for PEDV, and the PEDV-positive cases were mainly detected in winter. Then, we selected the SXSL strain as a representative strain to study the genetic and pathogenic characterization of PEDV pandemic strains in mid-west China. The recombination analysis showed that SXSL strain was a recombinant strain, and the major and minor parent strains of the recombination are CH/SCZJ/2018 strain and GDS48 strain, respectively. Complete genome sequencing and homology analysis showed that the S protein of SXSL strain contained multiple amino acid indels and mutations compared to the PEDV representative strains. Furthermore, we evaluated the effect of S protein on the infectivity and pathogenicity of PEDV by the PEDV reverse genetics system, and results showed that SXSL S protein increased the infectivity and pathogenicity of chimeric virus. Overall, our findings provided important information for understanding the roles of S protein in the prevalence of PEDV in mid-west China and developing vaccines based on PEDV pandemic strains.

Complete genomic sequence analysis and intestinal tissue localization of a porcine Kobuvirus variant in China.

Porcine kobuvirus (PKV) infection is very common in both healthy pigs and diarrhea pigs throughout the world. However, there is no proof that it causes diarrhea, and little is known about its role in diarrhea. There are only a few reports concerning porcine kobuvirus separation at present, which makes investigating its invasion and pathogenesis mechanisms difficult. This study sequenced the entire genome of a porcine kobuvirus strain termed "Wuhan2020" after it was isolated from intestinal tissue samples of healthy piglets. The analysis results revealed that it shared the most resemblance with the WUH1 strain (89.5%) and belonged to the same evolutionary branch as the Hungarian strain S-1-SUN. The PKV was located using the in situ hybridization (ISH) approach, which revealed that it was colonized in intestinal villus epithelial cells and lymphocytes in the Peyer's patch. In general, we analyzed the genetic evolution of PKV, discovered PKV susceptible cells and determined PKV localization in the intestine of infected pigs, providing a reference for future research.

Complete genome sequence of a novel mitovirus isolated from the fungus Fusarium equiseti causing potato dry rot.

In this study, a novel mitovirus was isolated from the fungus Fusarium equiseti causing potato dry rot and tentatively designated as "Fusarium equiseti mitovirus 1" (FeMV1). The full-length genome sequence of FeMV1 consists of 2,459 nucleotides with a predicted A + U content of 69.5%. Using the mold mitochondrial genetic code, an open reading frame (ORF) of 725 amino acids (aa) was predicted to encode an RNA-dependent RNA polymerase (RdRp). The RdRp protein contains six conserved motifs, with the highly conserved GDD in motif IV, and the 5'-untranslated region (UTR) and 3'-UTR of FeMV1 have the potential to fold into stem-loop secondary structures and a panhandle structure, both of which are typical characteristics of members of the family Mitoviridae. Results of a BLASTp search showed that the RdRp aa sequence of FeMV1 shared the highest sequence similarity with that of Fusarium poae mitovirus 2 (FpMV2) (76.84% identity, E-value = 0.0). Phylogenetic analysis based on the complete aa sequence of RdRp further suggested that FeMV1 is a new member of the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus associated with F. equiseti.

Comprehensive Genome and Plasmidome Analysis of Antimicrobial Resistant Bacteria in Wastewater Treatment Plant Effluent of Tokyo.

To characterize environmental antimicrobial resistance (AMR) in urban areas, extended-spectrum β-lactamase- (ESBL)/carbapenemase-producing bacteria (EPB/CPB, respectively) from urban wastewater treatment plant effluents in Tokyo were isolated on CHROMagar ESBL plate. Complete genome sequence analysis, including plasmids, indicated that 126 CTX-M-positive isolates (31%) were identified among the 404 obtained isolates. The CTX-M-9 group was predominant (n = 65, 52%), followed by the CTX-M-1 group (n = 44, 35%). Comparative genome analysis revealed that CTX-M-27-positive E. coli O16:H5-ST131-fimH41 exhibited a stable genome structure and clonal-global dissemination. Plasmidome network analysis revealed that 304 complete plasmid sequences among 85 isolates were grouped into 14 incompatibility (Inc) network communities (Co1 to Co14). Co10 consisted of primarily IncFIA/IncFIB plasmids harboring blaCTX-M in E. coli, whereas Co12 consisted primarily of IncFIA(HI1)/Inc FIB(K) plasmids harboring blaCTX-M, blaKPC, and blaGES in Klebsiella spp. Co11 was markedly located around Co10 and Co12. Co11 exhibited blaCTX-M, blaKPC, and blaNDM, and was mainly detected in E. coli and Klebsiella spp. from human and animal sources, suggesting a mutual role of Co11 in horizontal gene transfer between E. coli and Klebsiella spp. This comprehensive resistome analysis uncovers the mode of relational transfer among bacterial species, highlighting the potential source of AMR burden on public health in urban communities.

Analysis of complete chloroplast genome sequences and insight into the phylogenetic relationships of Ferula L

BackgroundFerula L. is one of the largest and most taxonomically complicated genera as well as being an important medicinal plant resource in the family Apiaceae. To investigate the plastome features and phylogenetic relationships of Ferula and its neighboring genera Soranthus Ledeb., Schumannia Kuntze., and Talassia Korovin, we sequenced 14 complete plastomes of 12 species. ResultsThe size of the 14 complete chloroplast genomes ranged from 165,607 to 167,013 base pairs (bp) encoding 132 distinct genes (87 protein-coding, 37 tRNA, and 8 rRNA genes), and showed a typical quadripartite structure with a pair of inverted repeats (IR) regions. Based on comparative analysis, we found that the 14 plastomes were similar in codon usage, repeat sequence, simple sequence repeats (SSRs), and IR borders, and had significant collinearity. Based on our phylogenetic analyses, Soranthus, Schumannia, and Talassia should be considered synonymous with Ferula. Six highly divergent regions (rps16/trnQ-UUG, trnS-UGA/psbZ, psbH/petB, ycf1/ndhF, rpl32, and ycf1) were also detected, which may represent potential molecular markers, and combined with selective pressure analysis, the weak positive selection gene ccsA may be a discriminating DNA barcode for Ferula species.ConclusionPlastids contain abundant informative sites for resolving phylogenetic relationships. Combined with previous studies, we suggest that there is still much room for improvement in the classification of Ferula. Overall, our study provides new insights into the plastome evolution, phylogeny, and taxonomy of this genus.

Complete Genome Sequence Analysis of Bacillus subtilis Bbv57, a Promising Biocontrol Agent against Phytopathogens.

Plant growth-promoting rhizobacteria (PGPR) are a group of root-associated beneficial bacteria emerging as one of the powerful agents in sustainable plant disease management. Among the PGPR, Bacillus sp. has become a popular biocontrol agent for controlling pests and the diseases of several crops of agricultural and horticultural importance. Understanding the molecular basis of the plant growth-promoting and biocontrol abilities of Bacillus spp. will allow us to develop multifunctional microbial consortia for sustainable agriculture. In our study, we attempted to unravel the genome complexity of the potential biocontrol agent Bacillus subtilis Bbv57 (isolated from the betelvine’s rhizosphere), available at TNAU, Coimbatore. A WGS analysis generated 26 million reads, and a de novo assembly resulted in the generation of 4,302,465 bp genome of Bacillus subtilis Bbv57 containing 4363 coding sequences (CDS), of which 4281 were functionally annotated. An analysis of 16S rRNA revealed its 100% identity to Bacillus subtilis IAM 12118. A detailed data analysis identified the presence of >100 CAZymes and nine gene clusters involved in the production of secondary metabolites that exhibited antimicrobial properties. Further, Bbv57 was found to harbor 282 unique genes in comparison with 19 other Bacillus strains, requiring further exploration.

Establishment of a polymerase chain reaction-based method for strain-level management of Enterococcus faecalis EF-2001 using species-specific sequences identified by whole genome sequences

In the development and manufacture of fermented foods, it is crucial to control and manage the bacterial species used in the products. We previously reported a complete genome sequence analysis of the Enterococcus faecalis EF-2001 strain that was used for supplements. By comparing this sequence to the publicly available complete genome sequence of E. faecalis strains, we were able to identify specific sequences of the EF-2001 strain. We designed primer sets to amplify these specific regions and performed a polymerase chain reaction (PCR). We confirmed that the DNA fragments were specifically amplified in the genome of the EF-2001 strain, but not those of other lactic acid bacteria (LABs) or strains of the same genus. Furthermore, these primers amplified DNA fragments even in genomic DNA extracted from heat-treated bacteria at 121°C and foods containing the EF-2001 strain. These results suggest that this method allows for simple and highly accurate identification of specific fermentation strains, such as LABs at the strain level, which will be useful for controlling the quality of fermented foods.

Naturally acquired feline bocavirus type 1 and 3 infections in cats with neurologic deficits.

Feline bocaviruses (FBoVs) have been recognized as novel feline pathogens associated with gastrointestinal diseases. Although bocavirus infections in humans and animals present a broad range of clinical symptoms including neurologic diseases, the neuropathology caused by FBoV infection in cats is unknown. This study aims to investigate the presence of bocavirus in the brain samples of 78 cats showing neurologic deficits and 41 healthy cats using polymerase chain reaction (PCR) and to present the pathological findings of FBoV infection in brain tissues. Only five (6.41%, five out of 78) cats with neurological deficit were FBoV positive on PCR screening and were characterized as FBoV-1 (four out of five) and FBoV-3 (one out of five) by sequencing. Among FBoV-positive cases, viral DNA were detected by PCR in the cerebrum and brain stem of all FBoV-positive cases and rarely detected in the cerebellum of some cases. Histologically, all FBoV-positive cases revealed a variety of inflammatory responses. Among these, 80% (four out of five cases) showed multifocal neuronal vacuolation, mainly found in the cerebrum and brain stem. Eosinophilic inclusion-like materials were found within the nuclei of glial cells in the FBoV-3-positive case. In situ hybridization revealed FBoV DNA in oligodendroglia and vacuolated neurons detected using dual labelling with Olig-2 and NeuN immunohistochemistry, respectively. Transmission electron microscopy confirmed the presence of FBoV-3 virions in the nuclei of glial cells. Apart from localization in brain tissues, the FBoV DNA were also detected in multiple lymph nodes (five out of five) and some intestines (two out of five) of such positive cases, suggesting both parenteral and enteral infections. Complete genome sequence analysis revealed genetic diversity of detected FBoV-1, which were closely related to the strains found in China and Hong Kong, while the detected FBoV-3 presented distant monophyletic clade to previously detected FBoV-3 sequences. The FBoVs, together, should be considered a neurotropic virus and a possible cause for neuronal vacuolation in cats with neurologic deficits.

GENOME SEQUENCE ANALYSIS, RT-PCR DIAGNOSTICS AND CONSTRUCTION OF VIRAL PROTEIN-EXPRESSION CASSETES FOR KAZAKH ISOLATE OF ORDINARY AND ANDEAN STRAINS OF POTATO VIRUS S

Potato virus S (PVS) belongs to the genus Carlavirus of family Betaflexiviridae with a positive-sense single-stranded RNA genome of 8.5 Kb. PVS is one of the most prevalent viruses of cultivated potatoes in Kazakhstan. Here we report phylogenetic analysis of complete genome sequences of Kazakh isolates of PVS from potato cultivars Fortuna and Ushkonyr, design of PVS strain-specific diagnostic PCR and construction of expression cassettes for conserved viral proteins from both isolates. The Fortuna and Ushkonyr isolates were found to share 79.9% nucleotide identity with each other and belong respectively to previously-defined ordinary and Andean strains of PVS. Based on analysis of conserved and variable regions of available PVS isolates, we designed primers for reverse transcription (RT)-duplex PCR to detect both strains in single and mixed infections and established their prevalence in Almaty region of Kazakhstan. Coding sequences of triple gene block proteins (25K, 12K, 7K), coat protein (34K) and cysteine-rich protein (11K) as well as methyltransferase, peptidase, helicase and RNA-dependent RNA polymerase domains of viral replicase were subcloned from both Fortuna and Ushkonyr genomic RNAs into the binary vector pBIN19 under the control of CaMV 35S promoter and nopaline synthetase terminator. These expression cassettes will be used to further investigate the biological properties and strain characteristics of viral proteins by their transient expression in plant cells and tissues or their stable expression in transgenic plants.

Complete genome sequence analysis of Paris alphapartitivirus 1: a novel member of the genus Alphapartitivirus infecting Paris polyphylla var. yunnanensis.

A novel double-stranded RNA (dsRNA) virus, tentatively named "Paris alphapartitivirus 1" (ParAPV1, OL960006-OL960007), was detected in Paris polyphylla var. yunnanensis plants exhibiting leaf chlorosis and shrinkage symptoms in Yunnan. Its complete genome sequence was determined using Illumina and Sanger sequencing. ParAPV1 has a bipartite genome that consists of dsRNA1 (1,917bp) encoding the viral RNA-dependent RNA polymerase (RdRp), and dsRNA2 (1,818bp) encoding the putative coat protein (CP). Sequence comparisons showed that the RdRp and CP of ParAPV1 are most similar to those of pear alphapartitivirus (PpPV2), with 69.97% and 54.21% amino acid sequence identities respectively. Phylogenetic analysis of the RdRp amino acid sequences of ParAPV1 and other partitiviruses showed that ParAPV1 cluster with viruses in a clade containing alphapartitiviruses, and that its closest known relatives are PpPV2 (BBA66577) and rose partitivirus (RoPV, ANQ45203S). Taken together, these results suggest that ParAPV1 should be regarded as a new member of genus Alphapartitivirus in the family Partitiviridae. This is the first report of a partitivirus infecting P. polyphylla var. yunnanensis.

Complete genome sequence and phylogenetic analysis of red seabream iridovirus isolated from a cage-cultured small yellow croaker (Larimichthys polyactis) in China.

Iridoviruses are emerging pathogens that are widespread in diverse environments and hosts. Numerous members of the family Iridoviridae are known to cause severe disease in freshwater and marine organisms. Here, we report the complete genome sequence and phylogenetic analysis of iridovirus strain LPIV-ZS-2021, isolated from a small yellow croaker (Larimichthys polyactis) in China. The genome sequence comprises 110,560 bp with a G+C content of 53.42%, has 104 putative open reading frames (ORFs), and shares the highest sequence similarity with red seabream iridovirus (RSIV) isolated in Japan (98.61%). Phylogenetic analysis revealed that it belongs to RSIV clade 1. This is the first fully sequenced RSIV genome from a small yellow croaker. The host range expansion of members of the genus Megalocytivirus warrants further attention to determine its potential economic and ecological impact.

Complete mitochondrial genome sequence and comparative analysis of the cultivated yellow nutsedge.

As a monocotyledonous plant in family Cyperaceae, yellow nutsedge (Cyperus esculentus L.) is unique in accumulating a substantial amount of oil in underground tubers and provides a model system for studying oil accumulation in nonseed tissues. However, no data on the mitochondrial and nuclear genome sequences of this species are available, which greatly limits our understanding of its evolutionary characteristics and some essential biological mechanisms. In the present study, we report the first complete mitochondrial genome sequence of the cultivated yellow nutsedge. The analysis of the genome showed that the yellow nutsedge mitochondrial genome is 1,002,696 bp in size and encodes 62 genes consisting of 36 protein-coding genes (PCGs), 20 transfer RNA (tRNA) genes, and six ribosomal RNA (rRNA) genes. Compared with other angiosperms, yellow nutsedge mitochondrial genome contains much higher percentage of noncoding sequences (95.36%). Sixteen plastid-derived fragments were identified to be strongly associated with mitochondrial genes including one intact plastid-related gene (ndhH). Comparative analysis with seven other sequenced plant mitochondrial genomes revealed that two syntenic gene clusters, rps3-rpl16 and rps12-nad3, are highly conserved in all plant mitochondrial genomes, and the mitochondrial genome of yellow nutsedge is more similar to those of monocotyledons in the gene order. Phylogenetic analysis based on 13 shared protein-encoding genes in eight plant species showed that yellow nutsedge is evolutionarily more closely related to monocotyledonary species. Overall, the species-specific features of the cultivated yellow nutsedge mitochondrial genome provide additional information for the evolutionary and comparative genomic studies in the yellow nutsedge and other Cyperus species of the Cyperaceae family.

Evolution and diversity of human adenoviruses isolated from patients with keratoconjunctivitis.

Human adenovirus type 8 is a highly contagious eye disease and is considered as the most common epidemic keratoconjunctivitis worldwide. The virus may alter the course of detection as mutations and recombination in surface antigens are associated with binding and pathogenesis in human adenovirus. The recognition of new recombinant human adenovirus has been based on sequencing of three genes, penton base, hexon and fiber. 50 suspected samples of ocular keratoconjunctivitis were selected over 6 months. Following DNA extraction from isolates positive for cytopathic effect in each well, the complete sequences of hexon, fiber, and penton regions were performed on the genome of human adenovirus isolates using PCR. The sequences of capsid genes, including hexon, fiber, and penton were assessed to observe the evidence of recombination at the molecular level using genetic tools. The results of nucleotide and amino acid sequence of 5/50 patients with epidemic keratoconjunctivitis positive for hypervariable region of hexon (132aa -449), hypervariable of knob fiber (183aa -362) and hypervariable penton (106aa -466) isolates showed nucleotide and amino acid identity of 98% and 99.41%, 99% and 100%, 95% and 99.72% with hexon, fiber and penton of human adenovirus 8 subtypes. The results of phylogenetic tree and Simplot of the entire sequences and hypervariable regions of isolated hexon, fiber and penton showed all the isolates of human adenovirus from Ahvaz, Iran, were clustered with human adenovirus 8A, B, E, P and J, subtypes isolated strains from different regions of the world. The results of this study revealed that the human adenovirus isolates from patients with epidemic keratoconjunctivitis were closed to human adenovirus 8A, B, E, P and J subtypes. To determine the emergence of new human adenovirus D8 subtypes strain, analysis of complete genome sequence of human adenovirus was required.

In vivo Trial of Bifidobacterium longum Revealed the Complex Network Correlations Between Gut Microbiota and Health Promotional Effects.

Complete genome sequence analysis of Bifidobacterium longum subsp. longum BCBL-583 isolated from a Korean female fecal sample showed no virulence factor or antibiotic resistance gene, suggesting human safety. In addition, this strain has oxygen and heat tolerance genes for food processing, and cholesterol reduction and mucin adhesion-related genes were also found. For in vivo evaluations, a high fat diet (HFD) mouse model was used, showing that BCBL-583 administration to the model (HFD-583) reduced the total cholesterol and LDL-cholesterol in the blood and decreased pro-inflammatory cytokines but increased anti-inflammatory cytokines, substantiating its cholesterol reduction and anti-inflammation activities. Subsequent microbiome analysis of the fecal samples from the HFD mouse model revealed that BCBL-583 administration changed the composition of gut microbiota. After 9 weeks feeding of bifidobacteria, Firmicutes, Actinobacteria, and Bacteroidetes increased, but Proteobacteria maintained in the HFD mouse models. Further comparative species-level compositional analysis revealed the inhibitions of cholesterol reduction-related Eubacterium coprostanoligenes and obesity-related Lactococcus by the supplementation of B. longum BCBL-583, suggesting its possible cholesterol reduction and anti-obesity activities. The correlation analysis of HFD-583 between the gut microbiota compositional change and cholesterol/immune response showed that Verrucomicrobia, Firmicutes, Actinobacteria, and Bacteroidetes may play an important role in cholesterol reduction and anti-inflammation. However, correlation analysis of Proteobacteria showed the reverse correlation in HFD-583. Interestingly, the correlation analysis of B. longum ATCC 15707 administration to HFD model showed similar patterns of cholesterol but different in immune response patterns. Therefore, this correlation analysis suggests that the microbial composition and inflammatory cytokine/total-cholesterol may be closely related in the administration of BCBL-583 in the HFD mice group. Consequently, BCBL-583 could be a good probiotic strain for gut health promotion through gut microbiota modulation.

Comparative analysis of complete chloroplast genome sequences of five endangered species and new insights into phylogenetic relationships of Paris

Paris L. genus has been a precious traditional herb for more than 2000 years in China. However, due to overexploitation and habitat destruction, Paris is threatened by extinction. Similar morphological features cause the classification of Paris species in dispute. The chloroplast (cp) genome approach has been used to investigate the evolution of Paris. However, some studies confirm that the cp genome may result in misleading relationships because of the length variation, gaps/indels deletion, and incorrect models of sequence evolution in concatenated datasets. Therefore, there is a high demand for a reconstructed phylogenetic relationship and developed genetic markers to conserve these species. Recent studies have demonstrated that the protein-coding genes could provide a better phylogenetic relationship in the phylogenetic investigation. In this study, the complete cp genomes of five species were characterized, and the length of five cp genomes ranges from 162,927 bp to 165,267 bp, covering 89 protein-coding genes, 38 tRNA, and eight rRNA. The analysis of the repeat sequences, codon usage, RNA-editing sites, and comparison of cp genomes shared a high degree of conservation. Based on the protein-coding genes, the phylogenetic tree confirmed Paris’s position in the order Melanthiaceae, providing maximum support for a sister relationship of the subgenera Paris sensu strict (Paris s.s.) with the Daiswa and Trillium. In addition, the molecular clock showed that subgenus Paris was inferred to have occurred at about 52.81 Mya, whereas subgenus Daiswa has originated at 24.56 Mya, which was consistent with the phylogenetic investigation. This study provided a valuable insight into the evolutionary dynamics of cp genome structure in the family Melanthiaceae, and it also contributes to the bioprospecting and conservation of Paris species.

Characterization and complete genome sequence analysis of a newly isolatedphage against Vibrio parahaemolyticus from sick shrimp in Qingdao, China.

Foodborne diseases have become a serious havoc, where antimicrobial resistance is throwing significant challenges on daily basis. With the increase of drug-resistant bacteria and food-borne infection associated with Vibrio parahaemolyticus, new and effective strategies were needed to control the emergence of vibriosis. Lytic bacteriophages come up as a promising way to resist the pathogenic population in various applications. In this study, a V. parahaemolyticus specific phage vB_VpS_PG28 was isolated from sewage in the seafood market. Results showed vB_VpS_PG28, is strictly a lytic bacteriophage and has a relatively large burst size of 103 plaque-forming units per infected cell. Comparative genomic and bioinformatic analyses proved that vB_VpS_PG28 is a new bacteriophage that had a homologous relation with Vibrio phages of family Siphoviridae, especially with phage VH2_2019, but transmission electron microscopy of vB_VpS_PG28 morphology characterized its morphology is similar to that of Myoviridae family. In silico analysis indicated that the vB_VpS_PG28 genome consists of 82712 bp (48.08% GC content) encoding 114 putative ORFs without tRNA,and any gene associated with resistance or virulence factors has not been found. The bacteriophage in the present study has shown significant outcomes in order to control bacterial growth under in vitro conditions. Thus, we are suggesting a beneficiary agent against foodborne pathogens. Further, to ensure the safe usage of phage oral toxicity testing is recommended.

Monitoring AMR in Campylobacter jejuni from Italy in the last 10 years (2011-2021): Microbiological and WGS data risk assessment.

Campylobacter jejuni is considered as the main pathogen in human food‐borne outbreaks worldwide. Over the past years, several studies have reported antimicrobial resistance (AMR) in C. jejuni strains. In Europe, the official monitoring of AMR comprises the testing of Campylobacter spp. from food‐producing animals because this microorganism is responsible for human infections and usually predominant in poultry. Food‐producing animals are considered to be a major source of campylobacteriosis through contamination of food products. Concerns are growing due to the current classification of C. jejuni by the WHO as a ‘high priority pathogen’ due to the emergence of resistance to multiple drugs such as those belonging to the fluoroquinolones, macrolides and other classes, which limits the treatment alternatives. Knowledge about the contributions of different food sources to gastrointestinal disease is fundamental to prioritise food safety interventions and to establish proper control strategies. Assessing the genetic diversity among Campylobacter species is essential to the understanding of their epidemiology and population structure. Using a population genetic approach and grouping the isolates into sequence types within different clonal complexes, it is possible to investigate the source of the human cases. The work programme was aimed for the fellow to assess the AMR of C. jejuni isolated from humans, poultry and birds from wild and urban Italian habitats. Given the public health concern represented by resistant pathogens in food‐producing animals and the paucity of data about this topic in Italy, the aim was to identify correlations between phenotypic and genotypic AMR and comparing the origin of the isolates. The work programme allowed the fellow to acquire knowledge, skills and competencies on the web‐based tools used by IZSAM to process the NGS data and perform bioinformatics analyses for the identification of epidemiological clusters, the study of AMR patterns in C. jejuni isolates, and the assessment of the human exposure to such AMR pathogens. Furthermore, the fellow became able to transfer the acquired knowledge through innovative web‐based didactical tools applied to WGS and clustering of specific food‐borne pathogens, with particular reference to C. jejuni. To achieve this objective, 2,734 C. jejuni strains isolated from domestic and wild animals and humans, during the period 2011–2021 were analysed. The resistance phenotypes of the isolates were determined using the microdilution method with EUCAST breakpoints, for the following antibiotics: nalidixic acid, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, streptomycin, tetracycline. The data were complemented by WGS data for each strain, uploaded in the Italian information system for the collection and analysis of complete genome sequence of pathogens isolated from animal, food and environment (GENPAT) developed and maintained at IZSAM; information like clonal complex and sequence type to understand the phylogenetical distance between strains according to their origins were also considered. This work underlines that a better knowledge of the resistance levels of C. jejuni is necessary, and mandatory monitoring of Campylobacter species in the different animal productions is strongly suggested.

Role of Diversity and Recombination in the Emergence of Chilli Leaf Curl Virus.

Chilli leaf curl virus (ChiLCV), (Genus Begomovirus, family Geminiviridae) and associated satellites pose a serious threat to chilli production, worldwide. This study highlights the factors accountable for genetic diversity, recombination, and evolution of ChiLCV, and associated chilli leaf curl alphasatellite (ChiLCA) and chilli leaf curl betasatellite (ChiLCB). Phylogenetic analysis of complete genome (DNA-A) sequences of 132 ChiLCV isolates from five countries downloaded from NCBI database clustered into three major clades and showed high population diversity. The dN/dS ratio and Tajima D value of all viral DNA-A and associated betasatellite showed selective control on evolutionary relationships. Negative values of neutrality tests indicated purified selection and an excess of low-frequency polymorphism. Nucleotide diversity (π) for C4 and Rep genes was higher than other genes of ChiLCV with an average value of π = 18.37 × 10−2 and π = 17.52 × 10−2 respectively. A high number of mutations were detected in TrAP and Rep genes, while ChiLCB has a greater number of mutations than ChiLCA. In addition, significant recombination breakpoints were detected in all regions of ChiLCV genome, ChiLCB and, ChiLCA. Our findings indicate that ChiLCV has the potential for rapid evolution and adaptation to a range of geographic conditions and could be adopted to infect a wide range of crops, including diverse chilli cultivars.

Biocontrol Effect of Bacillus subtilis YPS-32 on Potato Common Scab and Its Complete Genome Sequence Analysis.

Potato common scab is caused by Streptomyces, which resides in soil and has become a serious disease in potato planting areas worldwide. In this study, we obtained a Bacillus subtilis YPS-32 strain by natural screening, and atmospheric and room-temperature plasma (ARTP) mutagenesis and field trial results showed that B. subtilis YPS-32 has a control efficacy of 83.70% against potato common scab. The complete genome of B. subtilis YPS-32 was sequenced, and multiple genes related to the synthesis of antibiotics and plant growth promoters were detected. Based on the genomic information for B. subtilis YPS-32, the sfp gene-inactivated (related to the synthesis of secondary metabolites) mutant strain B. subtilis YPS-32Δsfp was constructed. Analysis of crude extract metabolites using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) techniques revealed that strain YPS-32 encodes antagonists, such as surfactin and fengycin, which have antimicrobial effects. This study clarifies the mode of action by which B. subtilis YPS-32 antagonizes Streptomyces scabies and provides a reference for further research on antibacterial genes in the future.

Complete Genome Sequencing and Comparative Analysis of <i>Citrobacter koseri</i> CKNJ, a Strain Isolated from a Patient with Endogenous Endophthalmitis

Citrobacter koseri (C. koseri) is an opportunistic pathogen that can cause a variety of diseases. Although the mortality rate of C. koseri infections is high, there is a paucity of clinical information. Furthermore, the genomic features of this species are poorly understood. Herein, we present a patient with endogenous endophthalmitis secondary to septicemia, and collected a C. koseri isolate, CKNJ, from the blood of the patient. Whole genome sequencing revealed that CKNJ harbors no plasmids and codes for 67 putative virulence factors. Whole genome single nucleotide polymorphism-based phylogenetic analysis revealed that the CKNJ strain was close to strains with the same isolation sites. Compared to the other sequenced C. koseri chromosomes, CKNJ contains several strain-variable regions, including one prophage and 2 large genomic islands. Sequencing of the first complete genome of a clinical strain from China should reinforce our understanding of the genomic features and pathogenicity of this invasive infection-causing C. koseri with clinical significance.