Multi-resistant ST 258 Klebsiella pneumoniae presenting a hypermucoviscous phenotype

Multi-resistant ST 258 Klebsiella pneumoniae presenting a hypermucoviscous phenotype | 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 Article Multi-resistant ST 258 Klebsiella pneumoniae presenting a hypermucoviscous phenotype Alessandra Beatriz dos Santos Rondon Souza, Felipe Ramos Pinheiro, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4854582/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 To characterize phenotypically and genotypically a strain of Klebsiella pneumoniae ST258 isolated from an immunosuppressed patient treated at a University Hospital in Brazil, it was performed Antimicrobial Susceptibility Testing, Analysis of the Ability to form Biofilm, Genome Sequencing and String Test. The sample was characterized as a non-biofilm-forming, multi-resistant and hypermucoviscous strain. Genomic analysis revealed the presence of resistance genes, virulence genes and plasmids. This is the first report of hypervirulent hypermucoviscous KPC ST258 in Rio de Janeiro, Brazil. KPC ST258 represents a significant global health threat due to the limited available treatment to fight these infections. This observation poses a threat to the community and hospital environments, highlighting the need for increased surveillance to detect these strains. Klebsiella pneumoniae ST258 Multi-resistant hypermucoviscous WGS Figures Figure 1 INTRODUCTION Klebsiella pneumoniae is a gram-negative, encapsulated, facultative anaerobic, rod-shaped bacterium from the Enterobacteriaceae family. It is an opportunistic pathogen that can cause urinary tract infections, bacteremia and pneumonia [ 1 ]. Is intrinsically resistant to ampicillin through the production of the enzyme β-lactamase, encoded by the blaSHV gene[ 2 ]. The WHO recognizes carbapenemase-producing K. pneumoniae (KPC) as a critical threat to public health [ 3 ]. MDR-hv Klebsiella spp. are both hypervirulent and resistant to multiple antimicrobials, in addition to undergoing an evolution that may lead to the emergence of phenotypically new strains [ 4 ]. They have diverse genetic origins, with cases reported, to date, in Asia (mainland China, Hong Kong, and Taiwan), Europe (France, Norway, United Kingdom, and Russia), Africa (Egypt) and America (Canada, United States and Brazil)[ 5 ]. The accumulation of mutations such as modifications in the quinolone resistance-determining region (QRDR mutations in gyr A and par C), changes in the porin gene (ompK36) and modifications leding to the overexpression of efflux pumps (OEP mutations: acr AB, oqx AB, ram A and rar A) has been reported in Klebsiella spp. MDR-hv with these mutations present resistance to several antimicrobials such as quinolones, carbapenems and tigecyclines [ 6 ]. Hypermucoviscous (HM) hypervirulent K. pneumoniae (hvKP) is prone to causing metastatic spread and life-threatening diseases. Initially described in the Asia-Pacific region, it is now identified in Western countries as well. The HM phenotype appears to enhance the virulence of hvKP strains and is often considered a substitute marker for improved virulence. This phenotype appears to depend on rmpA/rmpA2-associated enhanced production of polysaccharides and can frequently be identified by string-test [ 7 ]. Additionally, K. pneumoniae of sequence type 258 (ST258), known for producing carbapenemase (KPC), frequently infects hospitalized patients, leaving them with few therapeutic alternatives. As carbapenems represent the last line of defense against life-threatening bacterial infections, this poses a significant challenge for treatment [ 8 ]. RESULTS On September 2, 2020, a 35-year-old immunosuppressed male was admitted to a University Hospital in Niteroi City, Rio de Janeiro, Brazil. According to his medical records, the patient presented with complaints of fever, cough, dyspnea, and seizure. These signs and symptoms began on August 30, 2020, four days prior to hospitalization. Upon admittion to the ICU (day 1), a preliminary examination showed an oxygen saturation (SO2) > 95%, hemoglobin level of 11,7 g/dL, hematocrit of 35,6%, platelet count of 223 x 10 3 /mm 3 . The white blood cell counts tests displayed 6,0 x 10 3 /mm 3 of lymphocytes and 8.0 x 103/mm 3 of band (immature neutrophils). The patient tested negative for SARS-CoV-2 by RT-PCR five times. On September 14, 2020 (day 13), Acinetobacter baumannii was retrieved from tracheal aspirate and blood culture at the hospital’s clinical microbiology laboratory. On September 23, 2020 (day 21), blood tests showed a hemoglobin level of 6.1 g/dL and hematocrit of 19.0%, suggesting a case of anemia. Additionally, laboratory results showed 4 x 10 3 /mm 3 of lymphocytes, 4 x 10 3 /mm 3 of eosinophils, 4 x 10 3 /mm 3 of monocytes, 15 x 10 3 /mm 3 of band neutrophils. A Klebsiella pneumoniae was retrieved from the tracheal aspirate collected for the SARS-CoV-2 by RT-PCR surveillance test. K. pneumoniae colonies were retrieved from a MacConkey agar plate overnight incubation at 37°C. The colonies were glucose and lactose-positive, red, and dome-shaped with mucoid and stickiness aspects. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) further confirmed the Bacterial species identification. The Antimicrobial Sensitivity Test (AST) revealed resistance to Meropenem, Imipenem, Tazobactam, Cefazolin, Cefepime, Gentamicin, Amikacin, Tobramycin, Aztreonam, Ciprofloxacin, Levofloxacin, and Norfloxacin, which classified it as a multi-resistant strain. Analysis of Whole genome sequencing and assembly classified the sample as ST258 and revealed the presence of resistance genes against Aminoglycosides ( aph (3)-Ib/ aph (6)-Id/ rmt B/ aac (3)-IId/ aad A2/ aph (3)-Ia), ESBL ( fos A6/ bla CTX−M−14 / bla TEM−1B ), Macrolides ( mph (A)/ erm (42)), Chloramphenicol ( cat A1), Carbapenemase (bla KPC−2 ), Sulfonamides ( sul 2), Tetracycline ( tet (G)) and Trimetroprim ( dfr A12). The Virulence Finder Database (VFDB) revealed a total of 54 virulence genes, including acr A, acr B (efflux pump); ent A-F, fep A-D, and fep G (siderophore-iron uptake); fin A-H (Adherence – Type I fimbriae); mrk A-D, mrk F, mrk H-I (Adherence – Type III fimbriae) and iut A (Aerobactin- iron uptake). Plasmids were observed (ColRNAI, IncFIB(K), IncFII(K), and IncN_1) and the following mutations were identified PmrB: T246A, PmrB: R256G (deleterious), and MgrB truncation: 23/47. The isolate was not classified as biofilm-forming. However, it was String Test positive, exhibiting a dense filament greater than five millimeters upon touching the colony’s surface, indicating hypermucoviscosity (Fig. 1 ). The patient's medical records indicate that his death occurred on October 19, 2020. DISCUSSION Carbapenems play a crucial role in treating certain multidrug-resistant Gram-negative pathogens. The persistent spread of carbapenem-resistant bacteria, like ST258 K. pneumoniae, poses a significant worldwide health threat due to the scarcity of effective treatment options for these infections [ 7 ]. In a study carried out by Zafer and collaborators, the hypermucoviscous (HM) and hypervirulent phenotype was linked to the presence of IncHI1B plasmid associated with rmp A rmp A2, iut A, iro N, bla CTX−M−15 , bla OXA−48 , and bla NDM−1 , however, no sample was associated with the ST258 profile [ 11 ]. In this case report, a similar virulence and resistance profile ( rmp A rmp A2, iut A, iro N, bla CTX−M−14 , bla KPC−2 ) was found in an ST258 strain, corroborating the potential for transmission of resistance harboring the IncHI1B plasmid. Also, mutations in the chromosomal rcsA and rcsB genes, part of a signaling system regulating capsular biosynthesis, can also result in the HM phenotype. To our knowledge, this is the first report of hypervirulent hypermucoviscous Klebsiella pneumoniae ST258 in Rio de Janeiro, Brazil. The ST258 K. pneumoniae in this study exhibited a multidrug-resistance profile, including resistance to carbapenems and harboring one gene associated with this class of antimicrobial agents. This poses a threat, and surveillance to detect these strains must be considered. The precise mechanisms underlying the HM phenotype remain unknown in certain strains. METHODS Sample Klebsiella pneumoniae was retrieved from the tracheal aspirate of an immunosuppressed patient on the twenty-first day of hospitalization. Antimicrobial Susceptibility Testing (AST) Colonies were suspended in sterile saline solution until they reached 0.5 McFarland. Then, confluent sowing was carried out on Müeller-Hinton agar medium (Kasvi, Paraná, Brazil). Antimicrobial discs (amoxicillin/clavulanic acid 20ug/10ug, ceftazidime 30ug, cefepime 30ug, cefotaxime 30ug, ceftriaxone 30ug, meropenem 10ug, imipenem 10ug, aztreonam 30ug, ciprofloxacin 5ug, levofloxacin 5ug, amikacin 30ug, gentamicin 10ug, trimethoprim-sulfamethoxazole 1.25/23.75ug and tetracycline 30ug) were placed on the plate and incubated at 37 ºC for 18 hours. The reading was carried out by measuring the complete diameter of the inhibition halo (CLSI, 2023) [ 9 ]. Biofilm Growth Using Crystal Violet Assay : This test was performed according to[ 10 ] with some adaptations. 200 µL of the inoculum diluted in Tryptic Soy Broth (TSB, Sigma-Aldrich, Saint Louis, Massachusetts, USA), was added to each of the three wells of a 96-well flat-bottom polystyrene plate (NUNC - ThemoFisher Scientifc, Waltham, Massachusetts, USA), and incubated for 24h at 37 ºC. After that the contents of each well were removed and the wells were washed with phosphate-buffered saline (PBS). The plate was dried and stained with 200µL of crystal violet, followed by incubation at room Temperature (RT) for 15 minutes. The plate was washed, dried and 200µL of absolute alcohol was added to each well. After 30 minutes, the plates were read in an ELISA (Enzyme Linked Immuno Sorbent Assay) microplate reader at 560 nm. The cutoff point was defined by the equation: cutoff point = OD (Mean White) + (3x Standard Deviation (White)). The results will be interpreted as follows: Non-biofilm forming (0) - OD(a) ≤ cutoff point; Weak biofilm former (+) - cutoff point < OD(a) ≤ 2 x cutoff point; Moderate biofilm former (++) − 2 x cutoff point < OD(a) ≤ 4 x cutoff point; Strong biofilm former (+++) − 4 x cutoff point < OD(a). Any negative OD value must be presented as zero. String test The bacterial sample was cultured on McConkey agar plate (Kasvi, Paraná, Brazil) and incubated for 24 hours at 37°C. After bacterial growth, the formation of a viscous filament was verified by touching the surface of a colony with a sterile loop. The formation of a filament larger than 5 millimeters when touching the colony and moving the loop upwards caracterizes the strain as hypermucoviscous [ 7 ]. DNA Extraction and Next Generation Sequencing Bacterial DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) and quantified using the QuantiFluor ONE ds DNA system (Promega, Wisconsin, USA). The sequencing library was created using the Nextera DNA Flex (Illumina, California, USA), and was quantified by fluorometry using the QuantiFluor® ONE ds DNA system. After dilution, the library was sequenced using the NextSeq 1000/2000 P2 (200 Cycles) or NextSeq 2000 (Illumina, California, USA). Sequence analysis was carried out using the CABGen v1.09 platform [ 8 ] and The Virulence Finder Database (VFDB). Declarations ACKNOWLEDGEMENTS This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ (Projeto REDES: E26/211.554/2019; E-26/201.328/2021), and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brazil (CAPES) Finance Code 001. AVAILABILITY OF DATA AND MATERIALS Sequence data that support the findings of this study have been deposited in the BioSample Database in the National Center for Biotechnology Information’s with the primary accession code SAMN43145338 ETHICS APPROVAL AND CONSENT TO PARTICIPATE The study was approved by the ethical committee in Research from the Medicin Faculty to the Universidade Federal Fluminense (CEP – FM/UFF), being registered under CAAE number 26823614.2.0000.5243. The studies were conducted following the local legislation and institutional requirements. The participants provided their informed consent to participate in this study. ADITIONAL INFORMATIONS: CONFLICT OF INTEREST The authors declare no conflict of interest. COMPETING INTEREST The authors declare no competing interests. References Wang, G., Zhao, G., Chao, X., Xie, L. & Wang, H. The Characteristic of Virulence, Biofilm and Antibiotic Resistance of Klebsiella pneumoniae . Int. J. Environ. Res. Public Health . 2020;17:6278. Holt, K. E. et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae , an urgent threat to public health. Proc. Natl. Acad. Sci. U S A. 2015;112:E3574- 3581. Wyres, K. L. et al. Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae . PLoS Genet . 2019;15:e1008114. Russo, T. A. & Marr, C. M. Hypervirulent Klebsiella pneumoniae . Clin. Microbiol. Rev. 2019;32:e00001-19. Tang, M., Kong, X., Hao, J. & Liu J. Epidemiological Characteristics and Formation Mechanisms of Multidrug-Resistant Hypervirulent Klebsiella pneumoniae . Front. Microbiol . 2020;11:581543. Cheng, Y-H., et al . Tigecycline- non-susceptible hypervirulent Klebsiella pneumoniae strains in Taiwan. J. Antimicrob. Chemother . 2020;75:309–17. Le, MN-T. et al . Comprehensive Analysis of Bacteriocins Produced by the Hypermucoviscous Klebsiella pneumoniae Species Complex. Microbiol. Spectr. 2023;11:e0086323. Duré, F. M. et al. CABGen: A Web Application for the Bioinformatic Analysis of Bacterial Genomes. Frontiers in Microbiology [Internet]. 2022 [cited 2022 Nov 1];13. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2022.893474 CLSI. M100Ed33 | Performance Standards for Antimicrobial Susceptibility Testing, 33rd Edition [Internet]. Clinical & Laboratory Standards Institute. 2023 [cited 2023 Apr 28]. Available from: https://clsi.org/standards/products/microbiology/documents/m100/ Viksne, R., Racenis, K., Broks, R., Balode, A.O., Kise, L., Kroica, J. In Vitro Assessment of Biofilm Production, Antibacterial Resistance of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp. Obtained from Tonsillar Crypts of Healthy Adults. Microorganisms . 2023;11:258. Valiatti TB et al. Genomic Analysis of Klebsiella pneumoniae ST258 Strain Coproducing KPC-2 and CTX-M-14 Isolated from Poultry in the Brazilian Amazon Region. Antibiotics (Basel). 2022;11:1835. 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. 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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-4854582","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":350574534,"identity":"55ba9ddd-8ced-4e45-9be6-536cf0364195","order_by":0,"name":"Alessandra Beatriz dos Santos Rondon Souza","email":"","orcid":"","institution":"Laboratory of Molecular Epidemiology and Biotechnology, School of Pharmacy/Fluminense Federal University","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"Beatriz dos Santos 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Test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4854582/v1/65e57ac4c2668326bb1c5c0b.png"},{"id":66371760,"identity":"b87099d9-b9ca-4267-9a7c-7889a02b1872","added_by":"auto","created_at":"2024-10-11 04:47:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":816579,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4854582/v1/ac1f0769-0853-400f-a4d6-d0f2cce1091e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multi-resistant ST 258 Klebsiella pneumoniae presenting a hypermucoviscous phenotype","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e is a gram-negative, encapsulated, facultative anaerobic, rod-shaped bacterium from the Enterobacteriaceae family. It is an opportunistic pathogen that can cause urinary tract infections, bacteremia and pneumonia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Is intrinsically resistant to ampicillin through the production of the enzyme β-lactamase, encoded by the blaSHV gene[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The WHO recognizes carbapenemase-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e (KPC) as a critical threat to public health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMDR-hv \u003cem\u003eKlebsiella\u003c/em\u003e spp. are both hypervirulent and resistant to multiple antimicrobials, in addition to undergoing an evolution that may lead to the emergence of phenotypically new strains [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. They have diverse genetic origins, with cases reported, to date, in Asia (mainland China, Hong Kong, and Taiwan), Europe (France, Norway, United Kingdom, and Russia), Africa (Egypt) and America (Canada, United States and Brazil)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe accumulation of mutations such as modifications in the quinolone resistance-determining region (QRDR mutations in \u003cem\u003egyr\u003c/em\u003eA and \u003cem\u003epar\u003c/em\u003eC), changes in the porin gene (ompK36) and modifications leding to the overexpression of efflux pumps (OEP mutations: \u003cem\u003eacr\u003c/em\u003eAB, \u003cem\u003eoqx\u003c/em\u003eAB, \u003cem\u003eram\u003c/em\u003eA and \u003cem\u003erar\u003c/em\u003eA) has been reported in \u003cem\u003eKlebsiella\u003c/em\u003e spp. MDR-hv with these mutations present resistance to several antimicrobials such as quinolones, carbapenems and tigecyclines [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHypermucoviscous (HM) hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e (hvKP) is prone to causing metastatic spread and life-threatening diseases. Initially described in the Asia-Pacific region, it is now identified in Western countries as well. The HM phenotype appears to enhance the virulence of hvKP strains and is often considered a substitute marker for improved virulence. This phenotype appears to depend on rmpA/rmpA2-associated enhanced production of polysaccharides and can frequently be identified by string-test [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, \u003cem\u003eK. pneumoniae\u003c/em\u003e of sequence type 258 (ST258), known for producing carbapenemase (KPC), frequently infects hospitalized patients, leaving them with few therapeutic alternatives. As carbapenems represent the last line of defense against life-threatening bacterial infections, this poses a significant challenge for treatment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eOn September 2, 2020, a 35-year-old immunosuppressed male was admitted to a University Hospital in Niteroi City, Rio de Janeiro, Brazil. According to his medical records, the patient presented with complaints of fever, cough, dyspnea, and seizure. These signs and symptoms began on August 30, 2020, four days prior to hospitalization. Upon admittion to the ICU (day 1), a preliminary examination showed an oxygen saturation (SO2)\u0026thinsp;\u0026gt;\u0026thinsp;95%, hemoglobin level of 11,7 g/dL, hematocrit of 35,6%, platelet count of 223 x 10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe white blood cell counts tests displayed 6,0 x 10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e of lymphocytes and 8.0 x 103/mm\u003csup\u003e3\u003c/sup\u003e of band (immature neutrophils). The patient tested negative for SARS-CoV-2 by RT-PCR five times.\u003c/p\u003e \u003cp\u003eOn September 14, 2020 (day 13), \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e was retrieved from tracheal aspirate and blood culture at the hospital\u0026rsquo;s clinical microbiology laboratory. On September 23, 2020 (day 21), blood tests showed a hemoglobin level of 6.1 g/dL and hematocrit of 19.0%, suggesting a case of anemia. Additionally, laboratory results showed 4 x 10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e of lymphocytes, 4 x 10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e of eosinophils, 4 x 10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e of monocytes, 15 x 10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e of band neutrophils. A \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e was retrieved from the tracheal aspirate collected for the SARS-CoV-2 by RT-PCR surveillance test. \u003cem\u003eK. pneumoniae\u003c/em\u003e colonies were retrieved from a MacConkey agar plate overnight incubation at 37\u0026deg;C. The colonies were glucose and lactose-positive, red, and dome-shaped with mucoid and stickiness aspects. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) further confirmed the Bacterial species identification. The Antimicrobial Sensitivity Test (AST) revealed resistance to Meropenem, Imipenem, Tazobactam, Cefazolin, Cefepime, Gentamicin, Amikacin, Tobramycin, Aztreonam, Ciprofloxacin, Levofloxacin, and Norfloxacin, which classified it as a multi-resistant strain. Analysis of Whole genome sequencing and assembly classified the sample as ST258 and revealed the presence of resistance genes against Aminoglycosides (\u003cem\u003eaph\u003c/em\u003e(3)-Ib/ \u003cem\u003eaph\u003c/em\u003e(6)-Id/ \u003cem\u003ermt\u003c/em\u003eB/\u003cem\u003eaac\u003c/em\u003e(3)-IId/ \u003cem\u003eaad\u003c/em\u003eA2/ \u003cem\u003eaph\u003c/em\u003e(3)-Ia), ESBL ( \u003cem\u003efos\u003c/em\u003eA6/ bla\u003csub\u003eCTX\u0026minus;M\u0026minus;14\u003c/sub\u003e/ bla\u003csub\u003eTEM\u0026minus;1B\u003c/sub\u003e), Macrolides (\u003cem\u003emph\u003c/em\u003e(A)/ \u003cem\u003eerm\u003c/em\u003e(42)), Chloramphenicol (\u003cem\u003ecat\u003c/em\u003eA1), Carbapenemase (bla\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e), Sulfonamides (\u003cem\u003esul\u003c/em\u003e2), Tetracycline (\u003cem\u003etet\u003c/em\u003e(G)) and Trimetroprim (\u003cem\u003edfr\u003c/em\u003eA12). The Virulence Finder Database (VFDB) revealed a total of 54 virulence genes, including \u003cem\u003eacr\u003c/em\u003eA, \u003cem\u003eacr\u003c/em\u003eB (efflux pump); \u003cem\u003eent\u003c/em\u003eA-F, \u003cem\u003efep\u003c/em\u003eA-D, and \u003cem\u003efep\u003c/em\u003eG (siderophore-iron uptake); \u003cem\u003efin\u003c/em\u003eA-H (Adherence \u0026ndash; Type I fimbriae); \u003cem\u003emrk\u003c/em\u003eA-D, \u003cem\u003emrk\u003c/em\u003eF, \u003cem\u003emrk\u003c/em\u003eH-I (Adherence \u0026ndash; Type III fimbriae) and \u003cem\u003eiut\u003c/em\u003eA (Aerobactin- iron uptake). Plasmids were observed (ColRNAI, IncFIB(K), IncFII(K), and IncN_1) and the following mutations were identified PmrB: T246A, PmrB: R256G (deleterious), and MgrB truncation: 23/47.\u003c/p\u003e \u003cp\u003eThe isolate was not classified as biofilm-forming. However, it was String Test positive, exhibiting a dense filament greater than five millimeters upon touching the colony\u0026rsquo;s surface, indicating hypermucoviscosity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The patient's medical records indicate that his death occurred on October 19, 2020.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCarbapenems play a crucial role in treating certain multidrug-resistant Gram-negative pathogens. The persistent spread of carbapenem-resistant bacteria, like ST258 K. pneumoniae, poses a significant worldwide health threat due to the scarcity of effective treatment options for these infections [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a study carried out by Zafer and collaborators, the hypermucoviscous (HM) and hypervirulent phenotype was linked to the presence of IncHI1B plasmid associated with \u003cem\u003ermp\u003c/em\u003eA \u003cem\u003ermp\u003c/em\u003eA2, \u003cem\u003eiut\u003c/em\u003eA, \u003cem\u003eiro\u003c/em\u003eN, bla\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e, bla\u003csub\u003eOXA\u0026minus;48\u003c/sub\u003e, and bla\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e, however, no sample was associated with the ST258 profile [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this case report, a similar virulence and resistance profile (\u003cem\u003ermp\u003c/em\u003eA \u003cem\u003ermp\u003c/em\u003eA2, \u003cem\u003eiut\u003c/em\u003eA, \u003cem\u003eiro\u003c/em\u003eN, bla\u003csub\u003eCTX\u0026minus;M\u0026minus;14\u003c/sub\u003e, bla\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e) was found in an ST258 strain, corroborating the potential for transmission of resistance harboring the IncHI1B plasmid. Also, mutations in the chromosomal rcsA and rcsB genes, part of a signaling system regulating capsular biosynthesis, can also result in the HM phenotype.\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the first report of hypervirulent hypermucoviscous \u003cem\u003eKlebsiella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003epneumoniae\u003c/em\u003e ST258 in Rio de Janeiro, Brazil. The ST258 K. pneumoniae in this study exhibited a multidrug-resistance profile, including resistance to carbapenems and harboring one gene associated with this class of antimicrobial agents. This poses a threat, and surveillance to detect these strains must be considered. The precise mechanisms underlying the HM phenotype remain unknown in certain strains.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e \u003cstrong\u003eSample\u003c/strong\u003e \u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e was retrieved from the tracheal aspirate of an immunosuppressed patient on the twenty-first day of hospitalization.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAntimicrobial Susceptibility Testing (AST)\u003c/strong\u003e \u003cp\u003eColonies were suspended in sterile saline solution until they reached 0.5 McFarland. Then, confluent sowing was carried out on M\u0026uuml;eller-Hinton agar medium (Kasvi, Paran\u0026aacute;, Brazil). Antimicrobial discs (amoxicillin/clavulanic acid 20ug/10ug, ceftazidime 30ug, cefepime 30ug, cefotaxime 30ug, ceftriaxone 30ug, meropenem 10ug, imipenem 10ug, aztreonam 30ug, ciprofloxacin 5ug, levofloxacin 5ug, amikacin 30ug, gentamicin 10ug, trimethoprim-sulfamethoxazole 1.25/23.75ug and tetracycline 30ug) were placed on the plate and incubated at 37 \u0026ordm;C for 18 hours. The reading was carried out by measuring the complete diameter of the inhibition halo (CLSI, 2023) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofilm Growth Using Crystal Violet Assay\u003c/b\u003e: This test was performed according to[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] with some adaptations. 200 \u0026micro;L of the inoculum diluted in Tryptic Soy Broth (TSB, Sigma-Aldrich, Saint Louis, Massachusetts, USA), was added to each of the three wells of a 96-well flat-bottom polystyrene plate (NUNC - ThemoFisher Scientifc, Waltham, Massachusetts, USA), and incubated for 24h at 37 \u0026ordm;C. After that the contents of each well were removed and the wells were washed with phosphate-buffered saline (PBS). The plate was dried and stained with 200\u0026micro;L of crystal violet, followed by incubation at room Temperature (RT) for 15 minutes. The plate was washed, dried and 200\u0026micro;L of absolute alcohol was added to each well. After 30 minutes, the plates were read in an ELISA (Enzyme Linked Immuno Sorbent Assay) microplate reader at 560 nm. The cutoff point was defined by the equation: cutoff point\u0026thinsp;=\u0026thinsp;OD (Mean White) + (3x Standard Deviation (White)). The results will be interpreted as follows: Non-biofilm forming (0) - OD(a)\u0026thinsp;\u0026le;\u0026thinsp;cutoff point; Weak biofilm former (+) - cutoff point\u0026thinsp;\u0026lt;\u0026thinsp;OD(a)\u0026thinsp;\u0026le;\u0026thinsp;2 x cutoff point; Moderate biofilm former (++) \u0026minus;\u0026thinsp;2 x cutoff point\u0026thinsp;\u0026lt;\u0026thinsp;OD(a)\u0026thinsp;\u0026le;\u0026thinsp;4 x cutoff point; Strong biofilm former (+++) \u0026minus;\u0026thinsp;4 x cutoff point\u0026thinsp;\u0026lt;\u0026thinsp;OD(a). Any negative OD value must be presented as zero.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eString test\u003c/strong\u003e \u003cp\u003eThe bacterial sample was cultured on McConkey agar plate (Kasvi, Paran\u0026aacute;, Brazil) and incubated for 24 hours at 37\u0026deg;C. After bacterial growth, the formation of a viscous filament was verified by touching the surface of a colony with a sterile loop. The formation of a filament larger than 5 millimeters when touching the colony and moving the loop upwards caracterizes the strain as hypermucoviscous [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDNA Extraction and Next Generation Sequencing\u003c/strong\u003e \u003cp\u003eBacterial DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) and quantified using the QuantiFluor ONE ds DNA system (Promega, Wisconsin, USA). The sequencing library was created using the Nextera DNA Flex (Illumina, California, USA), and was quantified by fluorometry using the QuantiFluor\u0026reg; ONE ds DNA system. After dilution, the library was sequenced using the NextSeq 1000/2000 P2 (200 Cycles) or NextSeq 2000 (Illumina, California, USA). Sequence analysis was carried out using the CABGen v1.09 platform [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and The Virulence Finder Database (VFDB).\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Funda\u0026ccedil;\u0026atilde;o Carlos Chagas Filho de Amparo \u0026agrave; Pesquisa do Estado \u0026nbsp; do \u0026nbsp; Rio \u0026nbsp; \u0026nbsp;de \u0026nbsp; Janeiro \u0026nbsp; \u0026ndash; \u0026nbsp; \u0026nbsp;FAPERJ \u0026nbsp; (Projeto \u0026nbsp; REDES: \u0026nbsp; \u0026nbsp;E26/211.554/2019; \u0026nbsp; E-26/201.328/2021), and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brazil (CAPES) Finance Code 001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in the BioSample Database in the National Center for Biotechnology Information\u0026rsquo;s with the primary accession code SAMN43145338\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the ethical committee in Research from the Medicin Faculty to the Universidade Federal Fluminense (CEP \u0026ndash; FM/UFF), being registered under CAAE number 26823614.2.0000.5243. The studies were conducted following the local legislation and institutional requirements. The participants provided their informed consent to participate in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADITIONAL INFORMATIONS:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWang, G., Zhao, G., Chao, X., Xie, L. \u0026amp; Wang, H. The Characteristic of Virulence, Biofilm and Antibiotic Resistance of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cem\u003eInt. J. Environ. Res. Public Health\u003c/em\u003e. 2020;17:6278.\u003c/li\u003e\n \u003cli\u003eHolt, K. E. \u003cem\u003eet al.\u003c/em\u003e Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, an urgent threat to public health. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e U S A. 2015;112:E3574- 3581.\u003c/li\u003e\n \u003cli\u003eWyres, K. L. \u003cem\u003eet al.\u003c/em\u003e Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cem\u003ePLoS Genet\u003c/em\u003e. 2019;15:e1008114.\u003c/li\u003e\n \u003cli\u003eRusso, T. A. \u0026amp; Marr, C. M. Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cem\u003eClin. Microbiol.\u003c/em\u003e Rev. 2019;32:e00001-19.\u003c/li\u003e\n \u003cli\u003eTang, M., Kong, X., Hao, J. \u0026amp; Liu J. Epidemiological Characteristics and Formation Mechanisms of Multidrug-Resistant Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cem\u003eFront. Microbiol\u003c/em\u003e. 2020;11:581543.\u003c/li\u003e\n \u003cli\u003eCheng, Y-H., \u003cem\u003eet al\u003c/em\u003e. Tigecycline- non-susceptible hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strains in Taiwan. \u003cem\u003eJ. Antimicrob. Chemother\u003c/em\u003e. 2020;75:309\u0026ndash;17.\u003c/li\u003e\n \u003cli\u003eLe, MN-T. \u003cem\u003eet al\u003c/em\u003e. Comprehensive Analysis of Bacteriocins Produced by the Hypermucoviscous \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Species Complex. \u003cem\u003eMicrobiol. Spectr.\u003c/em\u003e 2023;11:e0086323.\u003c/li\u003e\n \u003cli\u003eDur\u0026eacute;, F. M. \u003cem\u003eet al.\u003c/em\u003e CABGen: A Web Application for the Bioinformatic Analysis of Bacterial Genomes. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e [Internet]. 2022 [cited 2022 Nov 1];13. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2022.893474\u003c/li\u003e\n \u003cli\u003eCLSI. M100Ed33 | Performance Standards for Antimicrobial Susceptibility Testing, 33rd Edition [Internet]. Clinical \u0026amp; Laboratory Standards Institute. 2023 [cited 2023 Apr 28]. Available from: https://clsi.org/standards/products/microbiology/documents/m100/\u003c/li\u003e\n \u003cli\u003eViksne, R., Racenis, K., Broks, R., Balode, A.O., Kise, L., Kroica, J. In Vitro Assessment of Biofilm Production, Antibacterial Resistance of \u003cem\u003eStaphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa,\u003c/em\u003e and \u003cem\u003eAcinetobacter\u003c/em\u003e spp. Obtained from Tonsillar Crypts of Healthy Adults. \u003cem\u003eMicroorganisms\u003c/em\u003e. 2023;11:258.\u003c/li\u003e\n \u003cli\u003eValiatti TB \u003cem\u003eet al.\u003c/em\u003e Genomic Analysis of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ST258 Strain Coproducing KPC-2 and CTX-M-14 Isolated from Poultry in the Brazilian Amazon Region. Antibiotics (Basel). 2022;11:1835.\u003c/li\u003e\n\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":"Klebsiella pneumoniae ST258, Multi-resistant, hypermucoviscous, WGS","lastPublishedDoi":"10.21203/rs.3.rs-4854582/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4854582/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo characterize phenotypically and genotypically a strain of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ST258 isolated from an immunosuppressed patient treated at a University Hospital in Brazil, it was performed Antimicrobial Susceptibility Testing, Analysis of the Ability to form Biofilm, Genome Sequencing and String Test. The sample was characterized as a non-biofilm-forming, multi-resistant and hypermucoviscous strain. Genomic analysis revealed the presence of resistance genes, virulence genes and plasmids. This is the first report of hypervirulent hypermucoviscous KPC ST258 in Rio de Janeiro, Brazil. KPC ST258 represents a significant global health threat due to the limited available treatment to fight these infections. This observation poses a threat to the community and hospital environments, highlighting the need for increased surveillance to detect these strains.\u003c/p\u003e","manuscriptTitle":"Multi-resistant ST 258 Klebsiella pneumoniae presenting a hypermucoviscous phenotype","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-11 04:38:57","doi":"10.21203/rs.3.rs-4854582/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":"33517d50-5739-4b49-b074-b87b91fe63a7","owner":[],"postedDate":"October 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-11T04:39:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-11 04:38:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4854582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4854582","identity":"rs-4854582","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}