Identification and toxicity evaluation of a Klebsiella pneumoniae strain isolated from diseased Eri silkworm (Samia ricini) with relevance to sericulture practices

Identification and toxicity evaluation of a Klebsiella pneumoniae strain isolated from diseased Eri silkworm (Samia ricini) with relevance to sericulture practices | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification and toxicity evaluation of a Klebsiella pneumoniae strain isolated from diseased Eri silkworm (Samia ricini) with relevance to sericulture practices Dimpi Moni Kalita¹, Sunayan Bardoloi², Sanghamitra Saharia¹, Sanjana Sharmin¹, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7259474/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The eri silkworm ( Samia ricini ) is an economically important non-mulberry silkworm extensively cultivated in Northeast India for its unique spun silk. Disease outbreaks among larvae, often of gut origin, pose a significant threat to sericulture in this tropical region. In the present study, a bacterial isolate (S1_4) was recovered from the gut of diseased S. ricini larvae and identified as Klebsiella pneumoniae through 16S rRNA gene sequencing and phylogenetic analysis. Pathogenicity was assessed via toxicity bioassays, revealing a time-dependent increase in mortality. LC₅₀ values were determined to be 8.45 × 10⁸, 2.20 × 10⁷, 7.58 × 10⁶, and 7.49 × 10⁴ CFU/ml at 24, 48, 72, and 96 hours post-infection, respectively. Infected larvae exhibited sepsis-like symptoms and progressive death. This is the first report implicating K. pneumoniae in the pathology of S. ricini , establishing a previously undocumented host–pathogen association. These findings highlight the importance of microbial surveillance and early disease detection in tropical sericulture and suggest that S. ricini may serve as a promising invertebrate model for studying bacterial virulence and host–pathogen interactions. 16S rRNA sequencing insect pathology Klebsiella pneumoniae LC50 Samia ricini sericulture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Sericulture is a traditional agro-industry in India that supports millions of rural livelihoods through silkworm rearing, cocoon processing, and silk production. India ranks second globally in raw silk output, with the Northeastern region recognized for cultivating all four major types of silkworms—mulberry, muga, eri and oak tasar (Gudimalla et al. 2020 ; Anusha et al. 2015 ; Lokeshwari & Shantibala 2010 ). Among these, the eri silkworm ( Samia ricini ) has been fully domesticated and is reared extensively in the region due to its ease of rearing, nutritional value, and production of high-quality spun silk (Kim et al. 2012 ; Meth & Gogoi 2016 ; MsangoSoko et al. 2020 ). However, frequent disease outbreaks continue to threaten eri silkworm productivity, often associated with microbial infections. As a poikilothermic insect, S. ricini is highly sensitive to changes in environmental parameters such as temperature, humidity, larval density, and leaf quality (Duraimurugan 2018 ). These factors can disrupt gut microbial balance and create conditions conducive to the emergence of opportunistic pathogens including bacteria, fungi, viruses, and protozoa (Douglas 2015 ; Gogoi et al. 2023). The silkworm gut hosts a complex microbial community, and its members can exist in symbiotic, facultative, or pathogenic relationships with the host (Kikuchi 2009 ; Prado and Almeida 2009 ; Tada et al. 2011 ). Understanding the shifts in gut microbiota during diseased conditions can provide valuable insights into insect health and immune responses (Dee Tan and Bautista 2022 ). While several studies have characterized gut-associated pathogens in Bombyx mori and Antheraea assamensis (Choudhury et al. 2002 ; Haloi et al. 2016 ), similar data for S. ricini remain limited. This study aims to isolate and identify disease-causing bacteria from the gut of infected S. ricini larvae and evaluate their pathogenic potential through toxicity assays. By uncovering gut-associated microbial threats in diseased eri silkworms, the research contributes to a deeper understanding of host–microbe interactions in non-mulberry silkworms and supports the development of microbial surveillance strategies for sustainable sericulture in tropical regions. Materials and Methods Silkworm sampling and bacterial isolation Fifth instar larvae of Samia ricini exhibiting disease symptoms were collected from the Government Eri Silkworm Grainage, Barduar, Assam (25.938879ºN, 91.468233ºE). The gut tissues were aseptically dissected and homogenized in sterile physiological saline (0.85% NaCl) using a mortar and pestle (Thakur et al. 2015 ). Homogenates were serially diluted and plated on nutrient agar (NA), followed by incubation at 37°C for 24 hours to allow bacterial colony development (Haloi et al. 2016 ). Culture purification and isolate selection Colonies exhibiting distinct morphology were sub-cultured repeatedly on NA plates until axenic cultures were obtained. Among these, a single isolate labeled S1_4 was selected based on consistency of colony morphology and growth, and stored under refrigerated conditions for further analysis. DNA extraction and 16S rRNA sequencing Genomic DNA was isolated from S1_4 using a CTAB-based extraction protocol as outlined by Moore et al. ( 2004 ), with minor modifications. Briefly, overnight cultures in LB broth were harvested by centrifugation, and the pellet was lysed using Tris-EDTA buffer containing RNase A, proteinase K, and SDS. Following CTAB precipitation and phenol–chloroform extraction, DNA was precipitated with isopropanol, washed in 70% ethanol, and resuspended in TE buffer. DNA quality was confirmed on 1% agarose gel. PCR amplification of the 16S rRNA gene was performed using universal primers (16S-F and 16S-R). Amplification products (~ 1500 bp) were purified and subjected to Sanger sequencing using the BDT v3.1 cycle sequencing kit and ABI 3730xl Genetic Analyzer. Forward and reverse reads were assembled into a consensus sequence using alignment software (Saharia et al. 2024 ). Phylogenetic analysis The resulting 16S rRNA sequence was queried against the NCBI GenBank database using BLASTn (Altschul et al. 1997 ). The top 10 high-scoring hits were aligned with the isolate sequence using ClustalW. A phylogenetic tree was constructed using the neighbor-joining algorithm with 1,000 bootstrap replicates in MEGA11 software (Tamura et al. 2013 ). Preparation of bacterial inocula To assess pathogenicity, bacterial suspensions were prepared from overnight cultures of the isolate in nutrient broth. Cultures were centrifuged (5,000 rpm, 10 min), and the pellets resuspended in phosphate-buffered saline (PBS). A series of 10-fold dilutions were made to achieve concentrations ranging from 1×10⁹ to 1×10¹ CFU/ml (Haloi et al. 2016 ). Lethal concentration (LC₅₀) assay Groups of ten healthy fifth-instar larvae were injected with 10 µL of each bacterial dilution using a sterile microsyringe. Each treatment was performed in triplicate. A PBS-injected control group was also maintained. Larval mortality was recorded at 24, 48, 72, and 96 hours post-injection. The data were used to calculate LC₅₀ values at each time point via probit analysis using IBM SPSS Statistics v25.0, following Finney’s method (Finney 1964 ). Regression curves were plotted in MS Excel with probit mortality (Y-axis) vs. log-transformed concentration (X-axis) (Saharia et al. 2024 ). Results Identification and molecular confirmation The isolate designated S1_4 formed opaque, creamy colonies with raised and circular margins on nutrient agar. Genomic DNA extracted from the culture yielded a single, prominent band (~ 1500 bp) upon amplification with 16S rRNA universal primers (Fig. 1 ). Sanger sequencing and BLAST analysis confirmed the isolate as Klebsiella pneumoniae , showing 96.44% identity to reference strain DSM 30104 (Table 1 ). The sequence was submitted to GenBank under accession number OR485703. Phylogenetic analysis using the neighbor-joining method placed the isolate in close association with other K. pneumoniae strains, supported by strong bootstrap values (Fig. 2 ). Table 1 Top 10 BLAST hits against the Standard(nr/nt) database used to construct the phylogenetic tree Description Max Score Total Score Query Cover E value Per. Ident Accession Klebsiella pneumoniae strain DSM 30104 2483 2483 98% 0 96.44% NR_117683.1 Klebsiella pneumoniae strain DSM 30104 2460 2460 98% 0 96.17% NR_117686.1 Klebsiella pneumoniae strain DSM 30104 2459 2459 99% 0 96.05% NR_036794.1 Klebsiella pneumoniae strain DSM 30104 2455 2455 98% 0 96.11% NR_117684.1 Klebsiella pneumoniae strain DSM 30104 2449 2449 98% 0 96.04% NR_117685.1 Klebsiella pneumoniae strain DSM 30104 2442 2442 98% 0 96.09% NR_117682.1 Klebsiella quasipneumoniae subsp. quasipneumoniae strain 01A030 2438 2438 96% 0 96.48% NR_134062.1 Klebsiella quasipneumoniae subsp. similipneumoniae strain 07A044 2435 2435 96% 0 96.48% NR_134063.1 Klebsiella pneumoniae subsp. rhinoscleromatis strain R-70 2427 2427 96% 0 96.66% NR_037084.1 Klebsiella pneumoniae strain NBRC 14940 2425 2425 96% 0 96.29% NR_113702.1 Toxicity symptoms and larval behavior Larvae injected with the S1_4 isolate exhibited progressive signs of infection, including reduced locomotion, cessation of feeding, and discoloration consistent with sepsis. These symptoms became more pronounced with increasing post-infection duration. In contrast, larvae in the control group (injected with PBS) remained active and showed no signs of illness or mortality. LC₅₀ analysis and mortality trends Probit analysis revealed a clear dose- and time-dependent relationship between bacterial concentration and larval mortality. The calculated LC₅₀ values decreased with time: 8.45 × 10⁸ CFU/ml at 24 hours, 2.20 × 10⁷ CFU/ml at 48 hours, 7.58 × 10⁶ CFU/ml at 72 hours, and 7.49 × 10⁴ CFU/ml at 96 hours. These results suggest increasing pathogenicity of the isolate over time. Regression plots showed strong linear relationships between log concentration and probit mortality for all time intervals (Figs. 3 – 6 ). The mortality percentages and probit values at each tested concentration are detailed in Tables 2 – 5 . No mortality was recorded in the control groups throughout the 96-hour observation period, confirming that the deaths in treated groups were specific to K. pneumoniae infection. Table 2 Data on probit value against test concentrations of K.pneumoniae on 5th instar eri silkworm. Mortality at 24 hrs was presented as mean of three replicates of silkworm larvae. CFU/ml log10 of concentration total exposed mortality at 24 hrs % mortality probit value Probability 0 Control 10 0 0 0 0.00 1×10 1 1 10 0 0 0 .001 1×10 2 2 10 0 0 0 .004 1×10 3 3 10 0 0 0 .011 1×10 4 4 10 0.33 3.3 3.12 .028 1×10 5 5 10 1 10 3.72 .063 1×10 6 6 10 1.33 13.3 3.87 .127 1×10 7 7 10 2 20 4.16 .227 1×10 8 8 10 3.33 33.3 4.56 .359 1×10 9 9 10 5.3 53 5.08 .511 Table 3 Data on probit value against test concentrations of K.pneumoniae on 5th instar eri silkworm. Mortality at 48 hrs was presented as mean of three replicates of silkworm larvae. CFU/ml log10 of concentration total exposed mortality at 48 hrs % mortality probit value Probability 0 Control 10 0 0 0 0 1×10 1 1 10 0 0 0 0.001 1×10 2 2 10 0 0 0 0.004 1×10 3 3 10 0 0 0 0.015 1×10 4 4 10 0.66 6.6 3.45 0.047 1×10 5 5 10 1.33 13.3 3.87 0.121 1×10 6 6 10 2.66 26.6 4.36 0.251 1×10 7 7 10 4.33 43.3 4.82 0.432 1×10 8 8 10 6 60 5.25 0.628 1×10 9 9 10 8 80 5.84 0.796 Table 4 Data on probit value against test concentrations of K.pneumoniae on 5th instar eri silkworm. Mortality at 72 hrs was presented as mean of three replicates of silkworm larvae. CFU/ml log10 of concentration total exposed mortality at 72 hrs % mortality probit value Probability 0 Control 10 0 0 0 0 1×10 1 1 10 0 0 0 .004 1×10 2 2 10 0 0 0 .015 1×10 3 3 10 0.33 3.3 3.12 .042 1×10 4 4 10 1 10 3.72 .099 1×10 5 5 10 2.33 23.3 4.26 .201 1×10 6 6 10 4 40 4.75 .347 1×10 7 7 10 5.33 53.3 5.08 .521 1×10 8 8 10 6.66 66.6 5.41 .691 1×10 9 9 10 8 80 5.84 .828 Table 5 Data on probit value against test concentrations of K.pneumoniae on 5th instar eri silkworm. Mortality at 96 hrs was presented as mean of three replicates of silkworm larvae. CFU/ml log10 of concentration total exposed mortality at 96 hrs % mortality probit value Probability 0 Control 10 0 0 0 0 1×10 1 1 10 0.33 3.3 3.12 .089 1×10 2 2 10 1.6 16 4.01 .159 1×10 3 3 10 2.6 26 4.36 .257 1×10 4 4 10 4.6 46 4.9 .381 1×10 5 5 10 5.6 56 5.15 .517 1×10 6 6 10 6.3 63 5.33 .652 1×10 7 7 10 8 80 5.84 .770 1×10 8 8 10 8.3 83 5.95 .861 1×10 9 9 10 9 90 6.28 .924 Discussion This study adds to the growing body of knowledge in insect pathology by reporting the isolation and identification of a disease-causing bacterial strain from the gut of the eri silkworm, Samia ricini . Using 16S rRNA gene sequencing, the bacterium was identified as Klebsiella pneumoniae , a species more commonly recognized for its opportunistic infections in clinical settings. Similar molecular approaches for identifying entomopathogenic bacteria have been employed in earlier studies on Antheraea assamensis and Bombyx mori (Choudhury et al. 2002 ; Feng et al. 2011 ; Chavshin et al. 2014 ). For instance, Choudhury et al. ( 2002 ) isolated multiple bacterial strains from diseased A. assamensis larvae, among which strain AC-3 ( Pseudomonas aeruginosa ) exhibited 100% larval mortality within 72 hours post-injection, establishing its virulence. In the present study, the identification of K. pneumoniae was further validated by phylogenetic analysis, confirming its taxonomic placement and evolutionary relatedness to other known strains. This integrative approach has been advocated by several researchers (Saharia et al. 2024 ; Devi et al. 2022 ) to provide a more robust basis for bacterial classification and to understand evolutionary relationships among gut-associated pathogens. While K. pneumoniae has previously been reported as pathogenic in other insect species such as Galleria mellonella and Spodoptera litura (Insua et al. 2013 ; Devi et al. 2022 ), this is the first study to demonstrate its pathogenicity in S. ricini . The infected larvae displayed sluggishness, cessation of feeding, and symptoms consistent with systemic infection—similar to those observed by Devi et al. ( 2022 ). Interestingly, the LC₅₀ value determined in this study (7.58 × 10⁶ CFU/ml at 72 hours) was markedly lower than that reported for S. litura (1.2 × 10⁹ CFU/ml), suggesting a higher susceptibility in S. ricini or possible strain-specific differences in virulence. He et al. ( 2019 ) also reported significant mortality in Trichoplusia ni upon exposure to K. pneumoniae , supporting the entomopathogenic potential of this species across lepidopteran hosts. Such variations in pathogenicity may arise from several factors, including differences in host physiology, immune competence, or microbial strain virulence. The progressive decline in LC₅₀ values over time further indicates a cumulative toxic effect or a possible evasion of host immune responses, leading to increased mortality. Similar time-dependent pathogenic effects have been documented in other insect–bacterium models, where the buildup of bacterial toxins or unchecked proliferation within the hemocoel contributes to host death (He et al. 2019 ). The results underscore the need to monitor gut-associated microbial communities in rearing environments, particularly under stress conditions like fluctuating temperature, humidity, and overcrowding — all of which may facilitate opportunistic infections. Given the dual identity of K. pneumoniae as both an entomopathogen and a clinically significant bacterium in humans, its isolation from a silkworm host raises important questions about its ecological role and also about cross-kingdom host adaptability and microbial evolution. It also highlights the possibility that silkworms could serve not only as alternative hosts but also as useful models for studying host–microbe interactions and microbial pathogenicity. This aligns with the increasing use of insects such as G. mellonella in studies of host–pathogen interactions and bacterial virulence (Tsai et al. 2016, Ménard et al. 2021 ). While the study successfully establishes the pathogenicity of K. pneumoniae in S. ricini , it has some limitations. The underlying virulence mechanisms of the bacterial strain—such as toxin production, adhesion properties, or immune modulation—were not explored. Environmental stressors such as temperature fluctuations, host density, and dietary changes, which are known to affect disease outcomes in silkworms, were also not assessed here. Future investigations could include transcriptomic or proteomic profiling of infected larvae, comparative genomics of the bacterial isolate, or in vivo tracking of bacterial proliferation to provide deeper insights into the infection process. This study provides novel insights into the entomopathogenic potential of K. pneumoniae in S. ricini , with implications for disease surveillance and microbial risk management in sericulture. The findings underscore the need for continued monitoring of gut microflora, especially in the context of emerging or opportunistic pathogens that may compromise silkworm health and productivity. Conclusion This study establishes Klebsiella pneumoniae as a previously undocumented entomopathogen of Samia ricini , with demonstrated virulence supported by decreasing LC₅₀ values over time. The findings highlight a significant microbial threat to eri silkworm health and productivity in tropical sericulture systems. By identifying a gut-associated bacterial pathogen and quantifying its lethality, this research provides critical baseline data for disease monitoring and risk assessment in non-mulberry silkworms. Given the economic and cultural importance of eri silkworm rearing in India and Southeast Asia, continued surveillance and investigation into host–pathogen interactions are essential for safeguarding silkworm health. Future studies should focus on the mechanisms of infection, host immune responses, and environmental risk factors to develop targeted, sustainable disease management strategies for silkworm farmers. Declarations Acknowledgment The authors are highly indebted to the advanced-level Institutional Biotech Hub and Department of Zoology, B.Borooah College, Guwahati, Assam for providing laboratory facilities for the research work. Author contributions D.K.: Conceptualisation, Investigation; methodology; writing– original draft; writing– review and editing. S.B.: conceptualisation, visualisation, supervision, writing– review and editing. S.S.: Software, methodology. S.S.: Resources, methodology. P.D.: Resources, methodology. All authors reviewed the manuscript. Conflicts of interest The authors have no competing interests to declare that are relevant to the content of this article. Funding sources No funding was received to assist with the preparation of this manuscript. Data availability All data generated and analyzed are included in this research article. Ethical statement Not applicable. No vertebrate animals or human participants were used in this study. Use of artificial intelligence (AI)-assisted technology The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI. Data availability The 16S rRNA gene sequence of the bacterial isolate has been submitted to the NCBI GenBank database under accession number OR485703. All other data generated or analyzed during this study are available from the corresponding author on reasonable request. 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Virulence 7(3):214–229. http://doi.org/10.1080/21505594.2015.1135289 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7259474","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":533787009,"identity":"60b907cd-df74-4465-9d08-5dff1cdb9255","order_by":0,"name":"Dimpi Moni 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University","correspondingAuthor":false,"prefix":"","firstName":"Sunayan","middleName":"","lastName":"Bardoloi²","suffix":""},{"id":533787014,"identity":"fac7b324-6f2c-44ee-a866-2238cb031800","order_by":2,"name":"Sanghamitra Saharia¹","email":"","orcid":"","institution":"Gauhati University","correspondingAuthor":false,"prefix":"","firstName":"Sanghamitra","middleName":"","lastName":"Saharia¹","suffix":""},{"id":533787016,"identity":"1640508c-7e51-4d68-8f7b-0432c9797e9d","order_by":3,"name":"Sanjana Sharmin¹","email":"","orcid":"","institution":"Gauhati University","correspondingAuthor":false,"prefix":"","firstName":"Sanjana","middleName":"","lastName":"Sharmin¹","suffix":""},{"id":533787017,"identity":"fc195ce3-50fe-4d22-8c93-e5fc588adb06","order_by":4,"name":"Papori Deka¹","email":"","orcid":"","institution":"Gauhati University","correspondingAuthor":false,"prefix":"","firstName":"Papori","middleName":"","lastName":"Deka¹","suffix":""}],"badges":[],"createdAt":"2025-07-31 07:53:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7259474/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7259474/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94367889,"identity":"6b546733-b754-4b3b-823c-3a99e2f459db","added_by":"auto","created_at":"2025-10-27 13:13:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":740693,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/b8dddfd528b6b9c04a1471b1.docx"},{"id":94367689,"identity":"25eede67-7f80-45fd-9e80-b57728c725a6","added_by":"auto","created_at":"2025-10-27 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13:12:24","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112811,"visible":true,"origin":"","legend":"","description":"","filename":"352a3f1cc9f74f8ba41e691a779ed1ce1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/eb259717ea479b2ca566066f.xml"},{"id":94368069,"identity":"86d441ca-fe13-4ffd-b2fb-7f4bdc71ddbf","added_by":"auto","created_at":"2025-10-27 13:13:52","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120860,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/bf3e8a8e9d2d978ef96ca887.html"},{"id":94368060,"identity":"e2b57b4d-e640-4dbe-b0ea-004a5c8e6afa","added_by":"auto","created_at":"2025-10-27 13:13:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73814,"visible":true,"origin":"","legend":"\u003cp\u003egDNA and 16S Amplicon QC data of S1_4\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/e0c8802f3411ec6ed3f0074e.png"},{"id":94367682,"identity":"bb5fe5ae-09ad-4e05-979a-e4351948b9ff","added_by":"auto","created_at":"2025-10-27 13:13:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36270,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of S1_4 (neighbour joining method) with its closely related species (downloaded from NCBI) based on 16s rRNA sequencing\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/11eb2b41c5a793c9b744db58.png"},{"id":94368035,"identity":"8e72cee2-a5c4-4f48-9c22-93145c2cf180","added_by":"auto","created_at":"2025-10-27 13:13:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":33953,"visible":true,"origin":"","legend":"\u003cp\u003eRegression plot of probit mortality and log concentration for \u003cem\u003eK. pneumoniae\u003c/em\u003e to \u003cem\u003eS. ricini\u003c/em\u003e 5th instar larvae for 24 hours\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/8f6345337bdf3bf18855ccbd.png"},{"id":94367522,"identity":"7c9d7077-67cf-4628-9a32-b690177aa522","added_by":"auto","created_at":"2025-10-27 13:12:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33430,"visible":true,"origin":"","legend":"\u003cp\u003eRegression plot of probit mortality and log concentration for \u003cem\u003eK. pneumoniae\u003c/em\u003e to \u003cem\u003eS. ricini\u003c/em\u003e 5th instar larvae for 48 hours\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/ffccad06b483e666c17836ac.png"},{"id":94367800,"identity":"c80edfef-ce07-44ec-a9e5-5c3eccac1b82","added_by":"auto","created_at":"2025-10-27 13:13:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33638,"visible":true,"origin":"","legend":"\u003cp\u003eRegression plot of probit mortality and log concentration for \u003cem\u003eK. pneumoniae\u003c/em\u003e to \u003cem\u003eS. ricini\u003c/em\u003e 5th instar larvae for 72 hours\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/772bc09dfd46f9b0c8c424f1.png"},{"id":94367886,"identity":"87807c5b-2a15-4dd1-8132-ca32b0a35c62","added_by":"auto","created_at":"2025-10-27 13:13:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":24269,"visible":true,"origin":"","legend":"\u003cp\u003eRegression plot of probit mortality and log concentration for \u003cem\u003eK. pneumoniae\u003c/em\u003e to \u003cem\u003eS. ricini\u003c/em\u003e 5th instar larvae for 96 hours\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/149259273db3ac68bb1b50a9.png"},{"id":109444448,"identity":"81738985-81dd-452a-adf1-1013ac121789","added_by":"auto","created_at":"2026-05-18 07:57:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":570263,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7259474/v1/4cc36d91-7185-4ceb-a9b6-b04fdc1c2b9d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification and toxicity evaluation of a Klebsiella pneumoniae strain isolated from diseased Eri silkworm (Samia ricini) with relevance to sericulture practices","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSericulture is a traditional agro-industry in India that supports millions of rural livelihoods through silkworm rearing, cocoon processing, and silk production. India ranks second globally in raw silk output, with the Northeastern region recognized for cultivating all four major types of silkworms\u0026mdash;mulberry, muga, eri and oak tasar (Gudimalla et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Anusha et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lokeshwari \u0026amp; Shantibala \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Among these, the eri silkworm (\u003cem\u003eSamia ricini\u003c/em\u003e) has been fully domesticated and is reared extensively in the region due to its ease of rearing, nutritional value, and production of high-quality spun silk (Kim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Meth \u0026amp; Gogoi \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; MsangoSoko et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, frequent disease outbreaks continue to threaten eri silkworm productivity, often associated with microbial infections. As a poikilothermic insect, \u003cem\u003eS. ricini\u003c/em\u003e is highly sensitive to changes in environmental parameters such as temperature, humidity, larval density, and leaf quality (Duraimurugan \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These factors can disrupt gut microbial balance and create conditions conducive to the emergence of opportunistic pathogens including bacteria, fungi, viruses, and protozoa (Douglas \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Gogoi et al. 2023).\u003c/p\u003e\u003cp\u003eThe silkworm gut hosts a complex microbial community, and its members can exist in symbiotic, facultative, or pathogenic relationships with the host (Kikuchi \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Prado and Almeida \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tada et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Understanding the shifts in gut microbiota during diseased conditions can provide valuable insights into insect health and immune responses (Dee Tan and Bautista \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While several studies have characterized gut-associated pathogens in \u003cem\u003eBombyx mori\u003c/em\u003e and \u003cem\u003eAntheraea assamensis\u003c/em\u003e (Choudhury et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Haloi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), similar data for \u003cem\u003eS. ricini\u003c/em\u003e remain limited.\u003c/p\u003e\u003cp\u003eThis study aims to isolate and identify disease-causing bacteria from the gut of infected \u003cem\u003eS. ricini\u003c/em\u003e larvae and evaluate their pathogenic potential through toxicity assays. By uncovering gut-associated microbial threats in diseased eri silkworms, the research contributes to a deeper understanding of host\u0026ndash;microbe interactions in non-mulberry silkworms and supports the development of microbial surveillance strategies for sustainable sericulture in tropical regions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eSilkworm sampling and bacterial isolation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFifth instar larvae of Samia ricini exhibiting disease symptoms were collected from the Government Eri Silkworm Grainage, Barduar, Assam (25.938879\u0026ordm;N, 91.468233\u0026ordm;E). The gut tissues were aseptically dissected and homogenized in sterile physiological saline (0.85% NaCl) using a mortar and pestle (Thakur et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Homogenates were serially diluted and plated on nutrient agar (NA), followed by incubation at 37\u0026deg;C for 24 hours to allow bacterial colony development (Haloi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCulture purification and isolate selection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eColonies exhibiting distinct morphology were sub-cultured repeatedly on NA plates until axenic cultures were obtained. Among these, a single isolate labeled S1_4 was selected based on consistency of colony morphology and growth, and stored under refrigerated conditions for further analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA extraction and 16S rRNA sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenomic DNA was isolated from S1_4 using a CTAB-based extraction protocol as outlined by Moore et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), with minor modifications. Briefly, overnight cultures in LB broth were harvested by centrifugation, and the pellet was lysed using Tris-EDTA buffer containing RNase A, proteinase K, and SDS. Following CTAB precipitation and phenol\u0026ndash;chloroform extraction, DNA was precipitated with isopropanol, washed in 70% ethanol, and resuspended in TE buffer. DNA quality was confirmed on 1% agarose gel.\u003c/p\u003e\u003cp\u003ePCR amplification of the 16S rRNA gene was performed using universal primers (16S-F and 16S-R). Amplification products (~\u0026thinsp;1500 bp) were purified and subjected to Sanger sequencing using the BDT v3.1 cycle sequencing kit and ABI 3730xl Genetic Analyzer. Forward and reverse reads were assembled into a consensus sequence using alignment software (Saharia et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe resulting 16S rRNA sequence was queried against the NCBI GenBank database using BLASTn (Altschul et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The top 10 high-scoring hits were aligned with the isolate sequence using ClustalW. A phylogenetic tree was constructed using the neighbor-joining algorithm with 1,000 bootstrap replicates in MEGA11 software (Tamura et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of bacterial inocula\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess pathogenicity, bacterial suspensions were prepared from overnight cultures of the isolate in nutrient broth. Cultures were centrifuged (5,000 rpm, 10 min), and the pellets resuspended in phosphate-buffered saline (PBS). A series of 10-fold dilutions were made to achieve concentrations ranging from 1\u0026times;10⁹ to 1\u0026times;10\u0026sup1; CFU/ml (Haloi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eLethal concentration (LC₅₀) assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGroups of ten healthy fifth-instar larvae were injected with 10 \u0026micro;L of each bacterial dilution using a sterile microsyringe. Each treatment was performed in triplicate. A PBS-injected control group was also maintained. Larval mortality was recorded at 24, 48, 72, and 96 hours post-injection. The data were used to calculate LC₅₀ values at each time point via probit analysis using IBM SPSS Statistics v25.0, following Finney\u0026rsquo;s method (Finney \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1964\u003c/span\u003e). Regression curves were plotted in MS Excel with probit mortality (Y-axis) vs. log-transformed concentration (X-axis) (Saharia et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIdentification and molecular confirmation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe isolate designated S1_4 formed opaque, creamy colonies with raised and circular margins on nutrient agar. Genomic DNA extracted from the culture yielded a single, prominent band (~\u0026thinsp;1500 bp) upon amplification with 16S rRNA universal primers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Sanger sequencing and BLAST analysis confirmed the isolate as \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, showing 96.44% identity to reference strain DSM 30104 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sequence was submitted to GenBank under accession number OR485703. Phylogenetic analysis using the neighbor-joining method placed the isolate in close association with other \u003cem\u003eK. pneumoniae\u003c/em\u003e strains, supported by strong bootstrap values (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTop 10 BLAST hits against the Standard(nr/nt) database used to construct the phylogenetic tree\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMax Score\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal Score\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eQuery Cover\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eE value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePer. Ident\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAccession\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella pneumoniae strain DSM 30104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2483\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2483\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.44%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_117683.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella pneumoniae strain DSM 30104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2460\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2460\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.17%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_117686.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella pneumoniae strain DSM 30104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2459\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2459\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.05%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_036794.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella pneumoniae strain DSM 30104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2455\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2455\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.11%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_117684.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella pneumoniae strain DSM 30104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2449\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2449\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.04%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_117685.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella pneumoniae strain DSM 30104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2442\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2442\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.09%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_117682.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella quasipneumoniae subsp. quasipneumoniae strain 01A030\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2438\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2438\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e96%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.48%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_134062.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella quasipneumoniae subsp. similipneumoniae strain 07A044\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2435\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2435\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e96%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.48%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_134063.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella pneumoniae subsp. rhinoscleromatis strain R-70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2427\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2427\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e96%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.66%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_037084.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKlebsiella pneumoniae strain NBRC 14940\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2425\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2425\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e96%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.29%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNR_113702.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eToxicity symptoms and larval behavior\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLarvae injected with the S1_4 isolate exhibited progressive signs of infection, including reduced locomotion, cessation of feeding, and discoloration consistent with sepsis. These symptoms became more pronounced with increasing post-infection duration. In contrast, larvae in the control group (injected with PBS) remained active and showed no signs of illness or mortality.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLC₅₀ analysis and mortality trends\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProbit analysis revealed a clear dose- and time-dependent relationship between bacterial concentration and larval mortality. The calculated LC₅₀ values decreased with time: 8.45 \u0026times; 10⁸ CFU/ml at 24 hours, 2.20 \u0026times; 10⁷ CFU/ml at 48 hours, 7.58 \u0026times; 10⁶ CFU/ml at 72 hours, and 7.49 \u0026times; 10⁴ CFU/ml at 96 hours. These results suggest increasing pathogenicity of the isolate over time. Regression plots showed strong linear relationships between log concentration and probit mortality for all time intervals (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe mortality percentages and probit values at each tested concentration are detailed in Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. No mortality was recorded in the control groups throughout the 96-hour observation period, confirming that the deaths in treated groups were specific to \u003cem\u003eK. pneumoniae\u003c/em\u003e infection.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eData on probit value against test concentrations of \u003cem\u003eK.pneumoniae\u003c/em\u003e on 5th instar eri silkworm. Mortality at 24 hrs was presented as mean of three replicates of silkworm larvae.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCFU/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003elog10 of concentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003etotal exposed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003emortality at 24 hrs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e% mortality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eprobit value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eProbability\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.011\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.028\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.063\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.127\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.227\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e33.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.359\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.511\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eData on probit value against test concentrations of \u003cem\u003eK.pneumoniae\u003c/em\u003e on 5th instar eri silkworm. Mortality at 48 hrs was presented as mean of three replicates of silkworm larvae.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCFU/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003elog10 of concentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003etotal exposed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003emortality at 48 hrs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e% mortality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eprobit value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eProbability\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.015\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.047\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.121\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.251\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e43.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.432\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.628\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.796\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eData on probit value against test concentrations of \u003cem\u003eK.pneumoniae\u003c/em\u003e on 5th instar eri silkworm. Mortality at 72 hrs was presented as mean of three replicates of silkworm larvae.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCFU/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003elog10 of concentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003etotal exposed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003emortality at 72 hrs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e% mortality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eprobit value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eProbability\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.015\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.042\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.099\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.201\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.347\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.521\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e66.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.691\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.828\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eData on probit value against test concentrations of \u003cem\u003eK.pneumoniae\u003c/em\u003e on 5th instar eri silkworm. Mortality at 96 hrs was presented as mean of three replicates of silkworm larvae.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCFU/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003elog10 of concentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003etotal exposed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003emortality at 96 hrs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e% mortality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eprobit value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eProbability\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.257\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.381\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.517\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.652\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.770\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.861\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.924\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study adds to the growing body of knowledge in insect pathology by reporting the isolation and identification of a disease-causing bacterial strain from the gut of the eri silkworm, \u003cem\u003eSamia ricini\u003c/em\u003e. Using 16S rRNA gene sequencing, the bacterium was identified as \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, a species more commonly recognized for its opportunistic infections in clinical settings. Similar molecular approaches for identifying entomopathogenic bacteria have been employed in earlier studies on \u003cem\u003eAntheraea assamensis\u003c/em\u003e and \u003cem\u003eBombyx mori\u003c/em\u003e (Choudhury et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Chavshin et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For instance, Choudhury et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) isolated multiple bacterial strains from diseased \u003cem\u003eA. assamensis\u003c/em\u003e larvae, among which strain AC-3 (\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e) exhibited 100% larval mortality within 72 hours post-injection, establishing its virulence.\u003c/p\u003e\u003cp\u003eIn the present study, the identification of \u003cem\u003eK. pneumoniae\u003c/em\u003e was further validated by phylogenetic analysis, confirming its taxonomic placement and evolutionary relatedness to other known strains. This integrative approach has been advocated by several researchers (Saharia et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Devi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) to provide a more robust basis for bacterial classification and to understand evolutionary relationships among gut-associated pathogens.\u003c/p\u003e\u003cp\u003eWhile \u003cem\u003eK. pneumoniae\u003c/em\u003e has previously been reported as pathogenic in other insect species such as \u003cem\u003eGalleria mellonella\u003c/em\u003e and \u003cem\u003eSpodoptera litura\u003c/em\u003e (Insua et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Devi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), this is the first study to demonstrate its pathogenicity in \u003cem\u003eS. ricini\u003c/em\u003e. The infected larvae displayed sluggishness, cessation of feeding, and symptoms consistent with systemic infection\u0026mdash;similar to those observed by Devi et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Interestingly, the LC₅₀ value determined in this study (7.58 \u0026times; 10⁶ CFU/ml at 72 hours) was markedly lower than that reported for \u003cem\u003eS. litura\u003c/em\u003e (1.2 \u0026times; 10⁹ CFU/ml), suggesting a higher susceptibility in \u003cem\u003eS. ricini\u003c/em\u003e or possible strain-specific differences in virulence. He et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) also reported significant mortality in \u003cem\u003eTrichoplusia ni\u003c/em\u003e upon exposure to \u003cem\u003eK. pneumoniae\u003c/em\u003e, supporting the entomopathogenic potential of this species across lepidopteran hosts.\u003c/p\u003e\u003cp\u003eSuch variations in pathogenicity may arise from several factors, including differences in host physiology, immune competence, or microbial strain virulence. The progressive decline in LC₅₀ values over time further indicates a cumulative toxic effect or a possible evasion of host immune responses, leading to increased mortality. Similar time-dependent pathogenic effects have been documented in other insect\u0026ndash;bacterium models, where the buildup of bacterial toxins or unchecked proliferation within the hemocoel contributes to host death (He et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe results underscore the need to monitor gut-associated microbial communities in rearing environments, particularly under stress conditions like fluctuating temperature, humidity, and overcrowding \u0026mdash; all of which may facilitate opportunistic infections. Given the dual identity of \u003cem\u003eK. pneumoniae\u003c/em\u003e as both an entomopathogen and a clinically significant bacterium in humans, its isolation from a silkworm host raises important questions about its ecological role and also about cross-kingdom host adaptability and microbial evolution. It also highlights the possibility that silkworms could serve not only as alternative hosts but also as useful models for studying host\u0026ndash;microbe interactions and microbial pathogenicity. This aligns with the increasing use of insects such as \u003cem\u003eG. mellonella\u003c/em\u003e in studies of host\u0026ndash;pathogen interactions and bacterial virulence (Tsai et al. 2016, M\u0026eacute;nard et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile the study successfully establishes the pathogenicity of \u003cem\u003eK. pneumoniae\u003c/em\u003e in \u003cem\u003eS. ricini\u003c/em\u003e, it has some limitations. The underlying virulence mechanisms of the bacterial strain\u0026mdash;such as toxin production, adhesion properties, or immune modulation\u0026mdash;were not explored. Environmental stressors such as temperature fluctuations, host density, and dietary changes, which are known to affect disease outcomes in silkworms, were also not assessed here. Future investigations could include transcriptomic or proteomic profiling of infected larvae, comparative genomics of the bacterial isolate, or in vivo tracking of bacterial proliferation to provide deeper insights into the infection process.\u003c/p\u003e\u003cp\u003eThis study provides novel insights into the entomopathogenic potential of \u003cem\u003eK. pneumoniae\u003c/em\u003e in \u003cem\u003eS. ricini\u003c/em\u003e, with implications for disease surveillance and microbial risk management in sericulture. The findings underscore the need for continued monitoring of gut microflora, especially in the context of emerging or opportunistic pathogens that may compromise silkworm health and productivity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study establishes \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e as a previously undocumented entomopathogen of \u003cem\u003eSamia ricini\u003c/em\u003e, with demonstrated virulence supported by decreasing LC₅₀ values over time. The findings highlight a significant microbial threat to eri silkworm health and productivity in tropical sericulture systems. By identifying a gut-associated bacterial pathogen and quantifying its lethality, this research provides critical baseline data for disease monitoring and risk assessment in non-mulberry silkworms. Given the economic and cultural importance of eri silkworm rearing in India and Southeast Asia, continued surveillance and investigation into host\u0026ndash;pathogen interactions are essential for safeguarding silkworm health. Future studies should focus on the mechanisms of infection, host immune responses, and environmental risk factors to develop targeted, sustainable disease management strategies for silkworm farmers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors are highly indebted to the advanced-level Institutional Biotech Hub and Department of Zoology, B.Borooah College, Guwahati, Assam for providing laboratory facilities for the research work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.K.: Conceptualisation, Investigation; methodology; writing\u0026ndash; original draft; writing\u0026ndash; review and editing. S.B.: conceptualisation, visualisation, supervision, writing\u0026ndash; review and editing. S.S.: Software, methodology. S.S.: Resources, methodology. P.D.: Resources, methodology. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received to assist with the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All data generated and analyzed are included in this research article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. No vertebrate animals or human participants were used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUse of artificial intelligence (AI)-assisted technology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The 16S rRNA gene sequence of the bacterial isolate has been submitted to the NCBI GenBank database under accession number OR485703. All other data generated or analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAltschul SF, Madden TL, Sch\u0026auml;ffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein data base search programs. Nucleic Acids Res 25(17):3389\u0026ndash;3402. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/25.17.3389\u003c/span\u003e\u003cspan address=\"10.1093/nar/25.17.3389\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnusha HG, Bhaskar RN, Earanna N (2015) Molecular Characterization of Pathogenic Bacteria Isolated from Flacherie Disease of Silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. 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Virulence 7(3):214\u0026ndash;229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1080/21505594.2015.1135289\u003c/span\u003e\u003cspan address=\"10.1080/21505594.2015.1135289\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"16S rRNA sequencing, insect pathology, Klebsiella pneumoniae, LC50, Samia ricini, sericulture","lastPublishedDoi":"10.21203/rs.3.rs-7259474/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7259474/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe eri silkworm (\u003cem\u003eSamia ricini\u003c/em\u003e) is an economically important non-mulberry silkworm extensively cultivated in Northeast India for its unique spun silk. Disease outbreaks among larvae, often of gut origin, pose a significant threat to sericulture in this tropical region. In the present study, a bacterial isolate (S1_4) was recovered from the gut of diseased \u003cem\u003eS. ricini\u003c/em\u003e larvae and identified as \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e through 16S rRNA gene sequencing and phylogenetic analysis. Pathogenicity was assessed via toxicity bioassays, revealing a time-dependent increase in mortality. LC₅₀ values were determined to be 8.45 \u0026times; 10⁸, 2.20 \u0026times; 10⁷, 7.58 \u0026times; 10⁶, and 7.49 \u0026times; 10⁴ CFU/ml at 24, 48, 72, and 96 hours post-infection, respectively. Infected larvae exhibited sepsis-like symptoms and progressive death. This is the first report implicating \u003cem\u003eK. pneumoniae\u003c/em\u003e in the pathology of \u003cem\u003eS. ricini\u003c/em\u003e, establishing a previously undocumented host\u0026ndash;pathogen association. These findings highlight the importance of microbial surveillance and early disease detection in tropical sericulture and suggest that \u003cem\u003eS. ricini\u003c/em\u003e may serve as a promising invertebrate model for studying bacterial virulence and host\u0026ndash;pathogen interactions.\u003c/p\u003e","manuscriptTitle":"Identification and toxicity evaluation of a Klebsiella pneumoniae strain isolated from diseased Eri silkworm (Samia ricini) with relevance to sericulture practices","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-24 21:13:54","doi":"10.21203/rs.3.rs-7259474/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56d8b26b-abb0-4eca-b8da-819c06cd7325","owner":[],"postedDate":"October 24th, 2025","published":true,"recentEditorialEvents":[{"type":"decision","content":"Withdrawn","date":"2026-05-18T07:42:10+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T07:56:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-24 21:13:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7259474","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7259474","identity":"rs-7259474","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}