{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T08:45:52Z","timestamp":1792572352215,"version":"4.5.0"},"update-to":[{"DOI":"10.12688\/f1000research.183746.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000}}],"reference-count":34,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100014538","name":"Lembaga Pengelola Dana Pendidikan","doi-asserted-by":"publisher","award":["59544\/IT3.D10\/PT.01.03\/P\/B\/2025"],"award-info":[{"award-number":["59544\/IT3.D10\/PT.01.03\/P\/B\/2025"]}],"id":[{"id":"10.13039\/501100014538","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"\n Endophytic bacteria within medicinal plants are a largely untapped reservoir of bioactive compounds and adaptive genetic traits.\n Piper crocatum<\/ns3:italic>\n (red betel), a Southeast Asian medicinal herb rich in flavonoids, alkaloids, and phenolics, offers a chemically selective niche for endophytes, yet the genomes of its microbial associates remain poorly characterised.\n Kluyvera ascorbata<\/ns3:italic>\n occurs as a plant-associated endophyte and an opportunistic human pathogen, and its chromosomal\n bla<\/ns3:italic>\n KLUA gene is the recognised progenitor of the clinically important CTX-M-2 \u03b2-lactamase sublineage. We report a high-quality draft genome of endophytic strain PCL31, isolated from surface-sterilised\n Piper crocatum<\/ns3:italic>\n tissues collected in Bogor, Indonesia. Whole-genome sequencing used the Oxford Nanopore PromethION platform, with\n de novo<\/ns3:italic>\n assembly in Flye, polishing in Medaka, and quality assessment by QUAST and BUSCO. The assembly spans 4,978,299\u00a0bp across eight contigs (N50 1.45 Mbp; GC content 54.26%; 100% BUSCO completeness,\n enterobacterales_odb10<\/ns3:italic>\n , n\u00a0=\u00a0440). Species identity was confirmed by digital DNA\u2013DNA hybridisation (dDDH 88.6% against the type strain\n K. ascorbata<\/ns3:italic>\n ATCC 33433) and average nucleotide identity (ANI 98.6% against reference genome NZ_CP096201.1). Annotation identified 4,579 protein-coding sequences, 22 ribosomal RNA, 88 transfer RNA, and 81 non-coding RNA genes, and no CRISPR\/Cas systems. antiSMASH 8.0 predicted four biosynthetic gene clusters (BGCs): a 53.8\u00a0kb non-ribosomal peptide synthetase (NRPS)\u2013metallophore cluster matching the conserved enterobactin locus (MIBiG similarity 0.83), plus three low-similarity clusters (\u2264 0.50) of uncertain novelty \u2014 a ribosomally synthesised and post-translationally modified peptide (RiPP)-like cluster, a terpene-precursor cluster, and an azole-containing RiPP. CARD-RGI profiling returned one Perfect hit (\n bla<\/ns3:italic>\n CTX-M-2) and 25 Strict hits, the latter dominated by intrinsic\n Enterobacteriaceae<\/ns3:italic>\n efflux and target-modification genes; phenotypic confirmation was not performed. This genome provides a resource for bioprospecting secondary metabolites from medicinal-plant endophytes and underscores the value of plant-associated isolates for biosafety and antimicrobial-resistance surveillance in opportunistic pathogens.\n <\/ns3:p>","DOI":"10.12688\/f1000research.183746.2","type":"journal-article","created":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T11:44:05Z","timestamp":1787312645000},"page":"1073","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["High-Quality Draft Genome Sequence of Endophytic Kluyvera Ascorbata\u00a0Strain PCL31 Isolated from Piper Crocatum (red betel)"],"prefix":"10.12688","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1475-4693","authenticated-orcid":false,"given":"Elizabeth","family":"Sidhartha","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Graduate School Bogor West Java IPB University"},{"name":"Department of Biomedicine, School of Health and Life Sciences Jakarta i3L University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2709-7370","authenticated-orcid":false,"given":"Rahadian","family":"Pratama","sequence":"additional","affiliation":[{"name":"Biomolecule Division, Department of Biochemistry Bogor West Java IPB University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ernst Richard","family":"Ng","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, School of Health and Life Sciences Jakarta i3L University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Irene Nathania","family":"Christiawan","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, School of Health and Life Sciences Jakarta i3L University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yeshaya","family":"Valentine","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, School of Health and Life Sciences Jakarta i3L University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rio","family":"Hermantara","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, School of Health and Life Sciences Jakarta i3L University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"I Made","family":"Artika","sequence":"additional","affiliation":[{"name":"Biomolecule Division, Department of Biochemistry Bogor West Java IPB University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Okti Nadia","family":"Poetri","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases and Public Health, School of Veterinary Medicine and Biomedical Sciences Bogor West Java IPB University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0480-2388","authenticated-orcid":false,"given":"Mega","family":"Safithri","sequence":"additional","affiliation":[{"name":"Bioanalysis Division, Department of Biochemistry Bogor West Java IPB University"},{"name":"Tropical Biopharmaca Research Center Bogor West Java IPB University"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2026,8,21]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"D690-D699","DOI":"10.1093\/nar\/gkac920","article-title":"CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database.","volume":"51","author":"B Alcock","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"e31573","DOI":"10.1016\/j.heliyon.2024.e31573","article-title":"A review on mechanisms and prospects of endophytic bacteria in biocontrol of plant pathogenic fungi and their plant growth-promoting activities.","volume":"10","author":"M Ali","year":"2024","journal-title":"Heliyon."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"W32-W38","DOI":"10.1093\/nar\/gkaf334","article-title":"antiSMASH 8.0: extended gene cluster detection capabilities and analyses of chemistry, enzymology, and regulation.","volume":"53","author":"K Blin","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref34","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2012.00110","article-title":"CTX-M Enzymes: Origin and Diffusion.","volume":"3","author":"R Cant\u00f3n","year":"2012","journal-title":"Frontiers in Microbiology."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"3895-3903","DOI":"10.1128\/AAC.02412-14","article-title":"In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing.","volume":"58","author":"A Carattoli","year":"2014","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"1203-1212","DOI":"10.1038\/s41592-023-01940-w","article-title":"CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning.","volume":"20","author":"A Chklovski","year":"2023","journal-title":"Nature Methods."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"W246-W251","DOI":"10.1093\/nar\/gky425","article-title":"CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins.","volume":"46","author":"D Couvin","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref6","doi-asserted-by":"publisher","DOI":"10.1093\/gigascience\/giab008","article-title":"Twelve years of SAMtools and BCFtools.","volume":"10","author":"P Danecek","year":"2021","journal-title":"GigaScience."},{"key":"ref7","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btad311","article-title":"NanoPack2: population-scale evaluation of long-read sequencing data.","volume":"39","author":"W De Coster","year":"2023","journal-title":"Bioinformatics."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"245-250","DOI":"10.1248\/bpb.b20-00726","article-title":"Anti-allergic Inflammatory Components from the Leaves of Piper crocatum Ruiz & Pav.","volume":"44","author":"Y Gong","year":"2021","journal-title":"Biol. 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Biotechnol."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"521","DOI":"10.3390\/metabo12060521","article-title":"Genome Features and AntiSMASH Analysis of an Endophytic Strain Fusarium sp. R1.","volume":"12","author":"Y Liu","year":"2022","journal-title":"Metabolites."},{"key":"ref53","doi-asserted-by":"publisher","first-page":"4647-4654","DOI":"10.1093\/molbev\/msab199","article-title":"BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes.","volume":"38","author":"M Manni","year":"2021","journal-title":"Molecular Biology and Evolution,"},{"key":"ref18","doi-asserted-by":"publisher","first-page":"20764","DOI":"10.1038\/s41598-023-47121-9","article-title":"Genome mining reveals novel biosynthetic gene clusters in entomopathogenic bacteria.","volume":"13","author":"W Meesil","year":"2023","journal-title":"Sci. Rep."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"2182","DOI":"10.1038\/s41467-019-10210-3","article-title":"TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy.","volume":"10","author":"J Meier-Kolthoff","year":"2019","journal-title":"Nat. Commun."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"170-182","DOI":"10.30997\/ijar.v4i2.293","article-title":"Clusterization of Red Betel Leaves (Piper Crocatum) from Various Regions in Indonesia Based on Secondary Metabolite Fingerprint Analysis and Cytotoxicity Values.","volume":"4","author":"E Purnama","year":"2023","journal-title":"Indonesian Journal of Applied Research (IJAR)."},{"key":"ref23","doi-asserted-by":"publisher","DOI":"10.1099\/mgen.0.000206","article-title":"MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies.","volume":"4","author":"J Robertson","year":"2018","journal-title":"Microbial Genomics."},{"key":"ref24","doi-asserted-by":"publisher","DOI":"10.1128\/spectrum.04054-23","article-title":"Re-updating the taxonomy of Kluyvera genus for a better understanding of CTX-M \u03b2-lactamase origin.","volume":"12","author":"M Rodriguez","year":"2024","journal-title":"Microbiol. Spectrum."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"9437","DOI":"10.48048\/tis.2025.9437","article-title":"Analysis of Bioactive Compounds Piper crocatum as Inhibitors of Acetylcholinesterase In Silico and In Vitro.","volume":"22","author":"M Safithri","year":"2025","journal-title":"Trends in Sciences."},{"key":"ref26","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2022.981355","article-title":"Advancement in the molecular perspective of plant-endophytic interaction to mitigate drought stress in plants.","volume":"13","author":"P Salvi","year":"2022","journal-title":"Front. Microbiol."},{"key":"ref27","doi-asserted-by":"publisher","DOI":"10.1099\/mgen.0.000685","article-title":"Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification.","volume":"7","author":"O Schwengers","year":"2021","journal-title":"Microbial Genomics."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"699-730","DOI":"10.1007\/s10482-022-01736-6","article-title":"Prospecting potential of endophytes for modulation of biosynthesis of therapeutic bioactive secondary metabolites and plant growth promotion of medicinal and aromatic plants.","volume":"115","author":"D Singh","year":"2022","journal-title":"Antonie Van Leeuwenhoek."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"548","DOI":"10.3390\/microorganisms12030548","article-title":"Mining Biosynthetic Gene Clusters of Pseudomonas vancouverensis Utilizing Whole Genome Sequencing.","volume":"12","author":"P Tamang","year":"2024","journal-title":"Microorganisms."},{"key":"ref31","doi-asserted-by":"publisher","DOI":"10.3389\/fpls.2023.1248319","article-title":"Contribution of endophytes towards improving plant bioactive metabolites: a rescue option against red-taping of medicinal plants.","volume":"14","author":"S Tsipinana","year":"2023","journal-title":"Front. Plant Sci."},{"key":"ref32","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2025.1558567","article-title":"Effects and molecular mechanism of endophytic elicitors on the accumulation of secondary metabolites in medicinal plants.","volume":"16","author":"Y Wang","year":"2025","journal-title":"Front. Microbiol."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1007\/s00294-025-01332-x","article-title":"The CRISPR-cas repertoire of Kluyvera ascorbata: insights from genomic data.","volume":"71","author":"C Yilmaz-\u00c7olak","year":"2025","journal-title":"Current Genetics."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"2836","DOI":"10.3390\/microorganisms11122836","article-title":"Genomic Characteristion of Opportunistic Pathogen Kluyvera Reveals a Novel CTX-M Subgroup.","volume":"11","author":"K Yu","year":"2023","journal-title":"Microorganisms."}],"container-title":["F1000Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/f1000research.com\/articles\/15-1073\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/15-1073\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/15-1073\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T08:34:08Z","timestamp":1792571648000},"score":1,"resource":{"primary":{"URL":"https:\/\/f1000research.com\/articles\/15-1073\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,21]]},"references-count":34,"URL":"https:\/\/doi.org\/10.12688\/f1000research.183746.2","relation":{"has-version":[{"id-type":"doi","id":"10.12688\/f1000research.183746.1","asserted-by":"subject"}],"has-review":[{"id-type":"doi","id":"10.5256\/f1000research.208553.r523808","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.208553.r527702","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.208553.r527702","asserted-by":"object"},{"id-type":"doi","id":"10.5256\/f1000research.208553.r523808","asserted-by":"object"}]},"ISSN":["2046-1402"],"issn-type":[{"value":"2046-1402","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,21]]},"assertion":[{"value":"Indexed","URL":"https:\/\/f1000research.com\/articles\/15-1073\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.202819.r503971, Mushtak T. S. Al-Ouqaili, Department of Microbiology, University of Anbar, College of Medicine, Ramadi City, Anbar Governorate, 03 Aug 2026, version 1, 1 approved with reservations","URL":"https:\/\/f1000research.com\/articles\/15-1073\/v1#referee-response-503971","order":0,"name":"referee-response-503971","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"Mega Safithri<\/b>; \nPosted: 13 Aug 2026<\/i>; We thank the reviewer for the careful and constructive assessment, and for the encouraging appraisal of the work. The comments have materially improved the manuscript. We have revised the text accordingly and respond point by point below; all changes are incorporated in Version 2.   Reviewer comment: \nThe discussion of Piper crocatum emphasizes its medicinal value but does not sufficiently explain how its phytochemical composition may shape endophytic genome evolution. Expanding this ecological rationale would strengthen the justification for whole-genome sequencing.<\/i> \u200b\u200b\u200b\u200b\u200b\u200b\u200b                            \nResponse:<\/b> We thank the reviewer for this helpful suggestion. We have expanded the Introduction to make this rationale explicit, noting that the rich phytochemistry of \nPiper crocatum<\/i> imposes a selective niche that may favour detoxification, efflux, and specialised metabolic traits in resident endophytes, thus providing a concrete justification for whole-genome characterisation. We have additionally clarified the appropriateness of characterizing a culturable isolate for downstream metabolomic and phenotypic validation on which bioprospecting and biosafety assessment depend. (Revised paragraph 2 of the Introduction.) Reviewer comment: \nThe manuscript links plant-associated bacteria with antimicrobial resistance dissemination through horizontal gene transfer. However, this argument would benefit from additional explanation regarding: ecological pathways connecting plant microbiomes and clinical settings, evidence supporting gene exchange, relevance of Kluyvera as an environmental reservoir of clinically important resistance genes.<\/i> \nOtherwise, the transition appears somewhat abrupt.<\/i> \nResponse: <\/b>We agree that this transition was underdeveloped and have substantially revised it. Rather than invoking a general connection between plant microbiomes and clinical settings, we now anchor the argument in the specific, mechanistically documented example most relevant to this organism, that is the CTX-M family of extended-spectrum \u03b2-lactamases. We now explain that the chromosomal \nbla<\/i>KLU genes of environmental, plant-associated \nKluyvera<\/i> spp. are the recognised progenitors of the CTX-M family, which has been mobilised onto plasmids by the insertion sequences ISEcp1 and ISCR1 and subsequently disseminated among clinical pathogens (Humeniuk et al., 2002; Cantón et al., 2012; Rodriguez & Gutkind, 2024). We further note that, in \nK. ascorbata<\/i> specifically, the chromosomal \nbla<\/i>KLUA gene corresponds to the plasmid-borne CTX-M-2 cluster (Rodriguez & Gutkind, 2024; Yu et al., 2023), which directly frames the CTX-M-2 determinant we report in this genome. This provides the concrete ecological pathway, the evidence of horizontal mobilisation, and the reservoir role of \nKluyvera<\/i> that the reviewer requested. We have been careful to state that the documented pathway concerns environmental \nKluyvera<\/i>, and that characterising endophytic representatives extends surveillance of this reservoir into the plant-internal compartment rather than implying that endophyte-to-clinic transfer has itself been demonstrated.  (Revised paragraph 3 of the Introduction.) Reviewer comment: \nThe introduction states that "no complete genome characterisation of an endophytic Kluyvera ascorbata from Piper crocatum has been reported", but this claim should be supported by a more comprehensive literature context. The authors should clearly distinguish their work from previous genome reports of K. ascorbata isolated from environmental, plant-associated, or clinical sources, explaining precisely what knowledge gap this study fills.<\/i> \nResponse: <\/b>We have qualified this statement and now distinguish our isolate from existing K. ascorbata genomes. Publicly available genomes derive largely from clinical or environmental sources (e.g. the type strain ATCC 33433 and reference strain SK, NZ_CP096201.1), whereas no genome of an endophytic \nK. ascorbata<\/i> from a tropical medicinal plant such as \nPiper crocatum<\/i> has been reported; this defines the specific gap our resource fills. (Revised final paragraph of the Introduction.) Reviewer comment: \nThe manuscript should explain why this genome will be valuable beyond the current study\u2014for example, comparative genomics, evolutionary studies, genome mining, natural product discovery, or future surveillance of environmental antimicrobial resistance.<\/i> \nResponse: <\/b>We now state that the genome supports comparative and evolutionary genomics of the \nKluyvera<\/i>\/CTX-M lineage, genome mining and natural-product discovery from medicinal-plant endophytes, and environmental AMR surveillance. (Added to the Introduction aims and echoed in the Conclusion.) Reviewer comment: \nThe manuscript does not explain why enrichment and bacterial isolation were performed at 37°C, which is considerably higher than the natural environmental temperature of Piper crocatum. This incubation temperature may preferentially recover mesophilic bacteria with potential clinical relevance while excluding other endophytic taxa. The authors should justify this choice or discuss its implications for isolate recovery.<\/i> \nResponse: <\/b>We agree this warrants comment. 37 °C was chosen to target culturable, fast-growing endophytes compatible with downstream high-molecular-weight DNA extraction and long-read sequencing, and reflects a deliberate focus on mesophilic, potentially clinically relevant taxa. We now acknowledge explicitly that this temperature biases recovery toward mesophiles and away from other endophytic taxa and frame the isolate as one representative of the culturable community rather than a survey of total endophytic diversity. (Added to Methods and noted as a limitation.) Reviewer comment: \nAlthough plating of the final rinse water is reported, the methodology would be stronger if additional evidence of successful surface sterilization were included (e.g., imprint method or molecular verification). The authors should discuss whether plating alone is sufficient to exclude epiphytic contamination<\/i>. \nResponse: <\/b>We thank the reviewer for this point. Surface sterilization was validated by plating an aliquot of the final rinse water, with no growth observed, as one of the standards, widely used culture-based controls in endophyte isolation. We agree that tissue imprinting or amplicon-based verification would provide more stringent confirmation of endophytic origin. However, as the tissue was fully processed during isolation, these controls cannot be applied retrospectively. We have retained an explicit statement of the rinse-plating control in the Methods, and we will adopt imprinting as an additional control in future isolations. Reviewer comment: \nThe authors describe DNA integrity assessment but do not report the acceptance criteria used before sequencing.<\/i> \nPlease specify: minimum DNA concentration, A260\/A280 ratio, A260\/A230 ratio, DNA quantity loaded for library preparation. These parameters improve reproducibility.<\/i> \nResponse: <\/b>We have specified the DNA acceptance criteria applied prior to library preparation, based on the manufacturer's recommended input requirements for the SQK-NBD114.24 kit (minimum concentration of 15 ng\/\u03bcl, A260\/280 of 1.8, A260\/230 of 2.0\u20132.2, and DNA amount of 400 ng). (Revised Methods, \u201cDNA extraction and sequencing.\u201d) Reviewer comment: \nThe sequencing workflow is comprehensive, but no information is provided regarding: total number of reads, total bases generated, mean read length, N50 read length, sequencing depth (coverage). These values are essential because they directly influence assembly quality.<\/i> \nResponse: <\/b>Most of these are already reported. The first paragraph of Results and Table 1 give 117,809 reads, 174.6 Mbp total, read N50 of 3,007 bp, and ~35× estimated coverage. We have added the mean read length of 1482 for completeness. Reviewer comment: \nThe manuscript states: "assemblies with >95% completeness and <5% contamination were considered high-quality." However, BUSCO evaluates completeness and duplication\u2014not contamination. Contamination is usually estimated using tools such as: CheckM, CheckM2, GUNC, BlobToolKit. The authors should either replace "contamination" with "duplication," or include an independent contamination assessment. This is one of the most important methodological issues.<\/i> \nResponse: <\/b>We thank the reviewer for catching this; the reviewer is correct that BUSCO reports completeness and duplication, not contamination. We have corrected error and now report an independent contamination estimate. Contamination was assessed with CheckM2 v1.1.0, yielding 0 % contamination and 100 % completeness, confirming a clean assembly. (Added to Methods and Table 1.) Reviewer comment: \nThe manuscript states: "Results were interpreted based on the established ANI threshold (>90%)." This threshold is incorrect for bacterial species delineation. Current consensus is: 95\u201396% ANI for species boundaries. The manuscript should revise this statement and cite current ANI standards.<\/i> \nResponse: <\/b>We thank the reviewer for this correction. We have revised the threshold to the current consensus of 95\u201396% ANI for species boundaries. Our value (98.6% against NZ_CP096201.1) lies well above this boundary, so the species assignment is unchanged. (Revised Methods, "Taxonomic and phylogenomic analysis.") Reviewer comment: \nOnly Medaka polishing was performed. Please clarify: whether multiple polishing rounds were conducted, whether polishing converged, why no additional polishing (e.g., Racon) was performed.<\/i> \nResponse: <\/b>We thank the reviewer for this comment. We have now clarified that a single round of Medaka was performed. Reads were base called with the Dorado super-accuracy model. With super-accuracy ONT reads, Medaka is the developer-recommended consensus step and Racon is no longer required for this workflow. (Revised Methods, \u201cSequence processing and assembly.\u201d) Reviewer comment: \nAlthough the Bakta version is reported, the manuscript should indicate: annotation mode, genetic code used, whether pseudogenes were predicted, whether default parameters were employed.<\/i> \nResponse: <\/b>We have added the requested annotation details to the Methods. Genome annotation was performed with Bakta v1.8.2 using the default annotation workflow and the light database (db-light). Specifically: (i) annotation was run in the standard (non-metagenome) mode; (ii) coding sequences were predicted using translation table 11; (iii) pseudogene detection and annotation were enabled by default (the --skip-pseudo-option was not used), so pseudogenes were predicted; and (iv) default parameters were used throughout. (Revised Methods, "Functional annotation and genome mining.") Reviewer comment: \nThe reported sequencing depth (~35×) is relatively modest for generating a high-quality Oxford Nanopore-only bacterial genome assembly. Although the BUSCO completeness is excellent (100%), the authors should discuss whether this sequencing depth may have contributed to the fragmented assembly (eight contigs) and whether additional sequencing could improve contiguity.<\/i> \nThe authors attribute fragmentation entirely to rRNA operons because the read N50 (~3 kb) is shorter than the operon length. However, this explanation remains speculative. Other factors may also contribute, including repetitive insertion sequences, prophages, tandem repeats, GC-rich regions. The authors should either provide assembly graph evidence supporting this explanation or moderate the wording.<\/i> \nResponse: <\/b>We have moderated the interpretation of the assembly fragmentation and now note that, while the rRNA operons are the principal likely cause, other repetitive elements (insertion sequences, prophages, tandem repeats) may also contribute. We have additionally addressed the sequencing-depth point: we now state that contiguity was limited primarily by read length rather than depth, that the ~35× coverage \u2014 though modest \u2014 was sufficient to recover the complete gene space (100% BUSCO completeness, 0% CheckM2 contamination), and that a more contiguous assembly would be achieved most effectively by increasing read length rather than depth alone (Added to first paragraph of Results.) Reviewer comment:  \nThe authors attribute fragmentation entirely to rRNA operons because the read N50 (~3 kb) is shorter than the operon length. However, this explanation remains speculative. Other factors may also contribute, including repetitive insertion sequences, prophages, tandem repeats, GC-rich regions. The authors should either provide assembly graph evidence supporting this explanation or moderate the wording.<\/i> \nResponse: <\/b>We have moderated the wording and now present rRNA-operon repeats as the most likely principal contributor while acknowledging that additional repetitive elements (insertion sequences, prophages, tandem repeats) may also limit contiguity. (Revised first paragraph of Results.) Reviewer comment: \nThe manuscript reports assembly statistics but provides little context. It would strengthen the Results if the assembly quality (genome size, GC content, contig number, N50) were briefly compared with published Kluyvera ascorbata genomes to demonstrate consistency.<\/i> \nResponse: <\/b>We have added a brief comparison confirming that our genome size (4.98 Mbp) and GC content (54.26%) are consistent with published \nK. ascorbata<\/i> genomes (5.1 Mbp with GC content of 54% for NCBI RefSeq NZ_CP096201.1, and 4.9 Mbp with GC content of 54.5% for ATCC 33433), supporting assembly integrity. (Added to Results.) Reviewer comment: \nThe statement "Low similarity may reflect either true biosynthetic novelty..." is speculative. Low similarity in antiSMASH can also result from fragmented clusters, incomplete assemblies, annotation differences, limited database representation. The manuscript should avoid implying novelty without experimental validation.<\/i> We agree and have reinforced the existing caveat. The revised text states that low antiSMASH similarity may equally arise from cluster fragmentation across contigs, annotation differences, under-representation of \nKluyvera<\/i>-associated clusters in current reference databases, or biosynthetic novelty, requiring further metabolomic validation. (Revised Results, "Biosynthetic gene clusters.")   Reviewer comment:  \nThe manuscript identifies 26 AMR determinants, yet most are regulatory or efflux-associated genes. The wording could lead readers to interpret these as confirmed resistance genes. The authors should emphasize that many identified loci are intrinsic housekeeping genes, regulatory genes, putative contributors to resistance, rather than acquired resistance determinants.<\/i> \nResponse: <\/b>We share this concern and had framed the Strict hits as "predominantly intrinsic \nEnterobacteriaceae<\/i> efflux systems and chromosomal target-modification genes whose contribution to clinical resistance phenotype cannot be inferred from sequence alone." We have strengthened this to state explicitly that most loci are intrinsic, regulatory, or putative contributors rather than acquired resistance determinants. (Revised Results, "Antimicrobial resistance genes.") Reviewer comment: \nThe manuscript states "Perfect hit was blaCTX-M-2..." Since Kluyvera species possess chromosomal blaKLU genes that are ancestral to CTX-M enzymes, the authors should clarify whether CARD annotated this locus directly as CTX-M-2, or whether it represents a homologous ancestral chromosomal \u03b2-lactamase.<\/i> \nThis distinction has evolutionary importance.<\/i> \nResponse: <\/b>We have now noted that CARD-RGI returned a Perfect hit (100% identity, full-length) to the CTX-M-2 model. Consistent with \nK. ascorbata<\/i> being the recognised chromosomal progenitor of the CTX-M-2 sublineage, we note that this chromosomally located locus is best interpreted in the context of the ancestral blaKLUA lineage rather than as an acquired plasmid-borne ESBL. (Revised Results.) Reviewer comment: \nPhenotypic antimicrobial susceptibility testing is mentioned only near the end. Since the Results emphasize antimicrobial resistance genes, the absence of MIC or susceptibility testing should be acknowledged immediately after presenting the CARD results.<\/i> \nResponse: <\/b>We have noted the phenotypic-testing limitation directly following the CARD results, stating that MIC\/susceptibility testing was outside the scope of this Genome Note and that predicted determinants are not phenotypically confirmed. (Results, "Antimicrobial resistance genes.") Reviewer comment: \nThe absence of CRISPR\/Cas systems is reported without interpretation. A brief statement regarding whether CRISPR absence is common in Kluyvera species would improve biological context.<\/i> \nResponse: <\/b>We have added the statement that CRISPR\/Cas-negative result is consistent with previous report of low distribution of CRISPR\/Cas systems in the Kluyvera genus, to provide biological context. Reviewer comment: \nThe final paragraph appropriately states that detailed interpretation is beyond the scope of the Genome Note. However, several earlier paragraphs already provide biological interpretation (e.g., novelty of BGCs, AMR implications). The manuscript should maintain a consistent balance between descriptive reporting and interpretation.<\/i> \nResponse: <\/b>We have harmonised the tone: interpretive statements (BGC novelty, AMR implications) are now consistently hedged and framed as prediction requiring validation, so that they align with the closing statement that detailed functional interpretation is beyond the scope of a Genome Note. (Revised throughout Results and Conclusion.) Reviewer comment: \nThe conclusion should be objective, with further perspective, or should add at least a few sentences about future study\/future perspective.<\/i> \nResponse: <\/b>We have revised the Conclusion to state findings objectively and added a forward-looking paragraph outlining future work: metabolomic validation of the predicted BGCs, phenotypic AST to confirm the predicted resistome, and comparative genomics across endophytic and clinical Kluyvera to trace CTX-M evolution. (Revised Conclusion.) Suggested references \nResponse: <\/b>We have expanded the relevant literature throughout the manuscript where it strengthens the argument. We are grateful for the reviewer's expertise and thorough evaluation, which has improved the rigour and clarity of the manuscript. We hope Version 2 addresses all the reservations raised.","URL":"https:\/\/f1000research.com\/articles\/15-1073\/v2#referee-comment-16925","order":1,"name":"referee-comment-16925","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.208553.r523808, Arun Prasanna Nagarajan, King Abdullah University of Science and Technology (KAUST), Thuwal, 08 Oct 2026, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/15-1073\/v2#referee-response-523808","order":2,"name":"referee-response-523808","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.208553.r527702, Francisco Callejas-Hern\u00e1ndez, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, 21 Oct 2026, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/15-1073\/v2#referee-response-527702","order":3,"name":"referee-response-527702","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work is funded by the Indonesian Endowment Fund for Education (LPDP) on behalf of the Indonesian Ministry of Higher Education, Science and Technology and managed under the EQUITY Program (Contract No: 59544\/IT3.D10\/PT.01.03\/P\/B\/2025) assigned to Mega Safithri. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}