Caracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en Colombia
ilustraciones, diagramas
- Autores:
-
Soto Guzmán, Fredi Giovanni
- Tipo de recurso:
- Fecha de publicación:
- 2022
- Institución:
- Universidad Nacional de Colombia
- Repositorio:
- Universidad Nacional de Colombia
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unal.edu.co:unal/84491
- Palabra clave:
- 570 - Biología::576 - Genética y evolución
Staphylococcus aureus
Antibióticos
antibiotics
Staphylococcus aureus meticilino sensible
Efecto inóculo
Cefazolina
Alotipo BlaZ
Colombia
Methicillin – sensitive Staphylococcus aureus
Inoculum effect
Cefazolin
blaZ Allotype
Colombia
- Rights
- openAccess
- License
- Atribución-NoComercial 4.0 Internacional
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| dc.title.spa.fl_str_mv |
Caracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en Colombia |
| dc.title.translated.eng.fl_str_mv |
Genomic characterization of methicillin susceptible Staphylococcus aureus with inoculum effect in Colombia |
| title |
Caracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en Colombia |
| spellingShingle |
Caracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en Colombia 570 - Biología::576 - Genética y evolución Staphylococcus aureus Antibióticos antibiotics Staphylococcus aureus meticilino sensible Efecto inóculo Cefazolina Alotipo BlaZ Colombia Methicillin – sensitive Staphylococcus aureus Inoculum effect Cefazolin blaZ Allotype Colombia |
| title_short |
Caracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en Colombia |
| title_full |
Caracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en Colombia |
| title_fullStr |
Caracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en Colombia |
| title_full_unstemmed |
Caracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en Colombia |
| title_sort |
Caracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en Colombia |
| dc.creator.fl_str_mv |
Soto Guzmán, Fredi Giovanni |
| dc.contributor.advisor.none.fl_str_mv |
Reyes Manrique, Jinnethe Cristina Reguero Reza, María Teresa Jesús |
| dc.contributor.author.none.fl_str_mv |
Soto Guzmán, Fredi Giovanni |
| dc.contributor.researchgroup.spa.fl_str_mv |
Unidad de Genética y Resistencia antimicrobiana - UGRA. Universidad El Bosque. Bogotá D.C. |
| dc.subject.ddc.spa.fl_str_mv |
570 - Biología::576 - Genética y evolución |
| topic |
570 - Biología::576 - Genética y evolución Staphylococcus aureus Antibióticos antibiotics Staphylococcus aureus meticilino sensible Efecto inóculo Cefazolina Alotipo BlaZ Colombia Methicillin – sensitive Staphylococcus aureus Inoculum effect Cefazolin blaZ Allotype Colombia |
| dc.subject.agrovoc.none.fl_str_mv |
Staphylococcus aureus |
| dc.subject.agrovoc.spa.fl_str_mv |
Antibióticos |
| dc.subject.agrovoc.eng.fl_str_mv |
antibiotics |
| dc.subject.proposal.spa.fl_str_mv |
Staphylococcus aureus meticilino sensible Efecto inóculo Cefazolina Alotipo BlaZ Colombia |
| dc.subject.proposal.eng.fl_str_mv |
Methicillin – sensitive Staphylococcus aureus Inoculum effect Cefazolin blaZ Allotype Colombia |
| description |
ilustraciones, diagramas |
| publishDate |
2022 |
| dc.date.issued.none.fl_str_mv |
2022-12 |
| dc.date.accessioned.none.fl_str_mv |
2023-08-08T17:21:48Z |
| dc.date.available.none.fl_str_mv |
2023-08-08T17:21:48Z |
| dc.type.spa.fl_str_mv |
Trabajo de grado - Maestría |
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info:eu-repo/semantics/masterThesis |
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info:eu-repo/semantics/acceptedVersion |
| dc.type.content.spa.fl_str_mv |
Text |
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http://purl.org/redcol/resource_type/TM |
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acceptedVersion |
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https://repositorio.unal.edu.co/handle/unal/84491 |
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Universidad Nacional de Colombia |
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Repositorio Institucional Universidad Nacional de Colombia |
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https://repositorio.unal.edu.co/ |
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https://repositorio.unal.edu.co/handle/unal/84491 https://repositorio.unal.edu.co/ |
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Universidad Nacional de Colombia Repositorio Institucional Universidad Nacional de Colombia |
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spa |
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spa |
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(2014) Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120 Brauner A., Fridman O., Gefen O., Balaban N.Q. (2016). Distinguishing between resistance, tolerance and persistance to antibiotic treatment. Nat Rev Microbiol. Apr;14(5): 320 – 30. doi: 10.1038/nrmicro.2016.34 Brook, I. (1989). Inoculum effect. Reviews in Infectious Diseases, 11(3), 361-368 Bryant R.E., Alford R.H. (1977). Unsuccessful treatment of staphylococcal endocarditis with cefazolin. JAMA. Feb 7; 237(6): 569 – 70. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. (2009). BLAST plus: architecture and applications. BMC Bioinformatics. 10:1 Carvajal LP, Rincón S, Echeverri AM, Porras J, Ríos R, Ordoñez KM, Seas C, Gómez- Villegas SI, Díaz L, Arias CA, Reyes J. (2020). Novel insights into the classification of staphylococcal β-lactamases in relation to the cefazolin inoculum effect. Antimicrobial Agents Chemotherapy. 64: e02511-19 Casadevall A., Pirofski L.A. (2010). On Virulence. Virulence. Jan – Feb; 1(1):2 Chong YP., Park SJ., Kim ES., Bang KM., Kim SH., Lee SO., Choi SH., Jeong JY., Woo JH., Kim YS. (2015) Prevalence of blaZ gene types and the cefazolin inoculum effect among methicillin-susceptible Staphylococcus aureus blood isolates and their association with multilocus sequence types and clinical outcome. European Journal of Clinical Microbiology and Infectious Diseases. Feb;34(2): 349-55. doi: 10.1007/s10096-014-2241-5. Creech CB, Wood JB, Thomsen I. (2016) Best Practices for Treatment of Invasive Methicillin – susceptible Staphylococcus aureus Infections: The Case of Oxacillin. Journal of the Pediatric Infectious Diseases Society. Dec;5(4):480 – 482. doi: 10.1093/jpids/piw052 Cui L, Isii T, Fukuda M, Ochiai T, Neoh H, Camargo ILB Da C, Watanabe Y, Shoji M, Hiramatsu K. (2010). An RpoB Mutation Confers Dual Heteroresistance to Daptomycin and Vancomycin in S. aureus. Antimicrobial Agents and Chemotherapy. 54: 5222–33 D ́haeseleer P . (2006). What are DNA sequence motifs? Nature Biotechnology. Apr;24(4):423-5 Damon HA, Soussy CJ, Courvalin P. (1998). Characterization of Mutations in the rpoB Gene That Confer Rifampin Resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. Oct;42(10):2590-4 Edgar RC. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acid Research 32:1792–1797. Eisenberg E., Levanon E.Y. (2013). Human Housekeeping genes, revisited. Trends Genet. Oct;29(10): 569 - 74 Fildes, P. (1940). The mechanism of the anti-bacterial action of mercury. British Journal of Experimental Pathology, 21(2), 67 Foster TJ. (2017). Antibiotic resistance in Staphylococcus aureus. Current status and future prospects. FEMS Microbioloby Reviews. May 1;41(3):430-449 Fuda CC, Fisher JF., Mobashery A. (2005) b-lactam resistance in Staphylococcus aureus: the adaptative resistance of a plastic genome. Cellular Molecular Life Sciences. 62. 2617- 2633. Goldstein PB. (2014). Resistance to rifampicin: a review. The Journal of Antibiotics. 67, 625– 630 Green ER, Mecsas J, (2016). Bacterial secretion systems: an overview. Microbiology Spectrum. 4(1): VMBF.0012-2015. Horswill AR, Jenul C. (2018). Regulation of Staphylococcus aureus virulence. Microbiology Spectrum. 2018 February; 6(1): doi: 10.1128/microbiolspec.GPP3-0031. Jarraud S, Mougel C, Thiolouse J, Meugnier H, Forey F, Lina G, Nesme X, Ettiene J, Vandenesch F. (2002). Relationships between Staphylococcus aureus Genetic Background, virulence factors, agr Groups (alleles) and Human disease. Infection and Immunity. Feb;70(2):632 – 41.doi: 10.1128/IAI.70.2.631-641.2002 Jung N., Rieg S. (2018). Essentials in the management of S. aureus bloodstream infections. Infection. Aug: 46(4): 441-442. Kaase M, Lenga S, Friederich S, Szabados F, Sakinc T, Kleine B, Gatermann SG. (2008). Comparison of phenotypic methods for penicillinase detection in Staphylococcus aureus. Clinical Microbiology and Infection. Jun;14(6): 614-6. Kariyone, K., Harada, H., Kurita, M., & Takano, T. (1970). Cefazolin, a new semisynthetic cephalosporin antibiotic. I. Journal of Antibiotics, 23(3), 131-136 Keinhorster D, George SE, Weindenmaier C, Wolz C. (2019). Function and regulation of Staphylococcus aureus wall teichoic acids and capsular polysaccharides. International Journal of Medical Microbiology. Sep;309(6):151333 Kirby WM. (1944). Extraction of a highly potent penicillin inactivator from penicillin resistant Staphylococci. Science. Jun 2;99(2579): 452 – 3. Llarrul, L., Prorok M., Mobashery S. (2010). Binding of the Gene Repressor BlaI to the bla Operon in Methicillin-Resistant Staphylococcus aureus. Biochemistry, 49, 7975 – 7977 Lenhard, J. R., & Bulman, Z. P. (2019). Inoculum effect of β-lactam antibiotics. Journal of Antimicrobials and Chemotherapy, 74(10), 2825-2843 Lee S, Choe PG, Song KH, Park SW, Kim HB., Kim NJ, Kim EC, Park WB, Oh M. (2011). Is cefazolin Inferior to Nafcillin for Treatment of Methicillin – susceptible Staphylococcus aureus bacteriemia? Antimicrobial Agents and Chemotherapy. Nov;55(11): 5122-6. Doi:10.1128/AAC.00485-11 Lee OS, Lee S, Park S, Lee JE, Lee SH. (2019). The cefazolin inoculum effect and the presence of type A blaZ Gene according to agr Genotype in Methicillin-susceptible Staphylococcus aureus bacteremia. Infections and Chemotherapy. Dec; 51(4): 376-385 Levy S., Marshall B. (2004) Antibacterial resistance worldwide: causes, challenges and responses. Nature Medicine. Dec;10(12 Suppl): S122-9. Liu B, Zheng D, Jin Q, Chen L, Yang J. (2019). VFDB 2019: A comparative pathogenomic platform with an interactive web interface. Nucleic Acids Research. 47:D687–D692 Livorsi DJ, Crispell E, Satola SW, Burd EM, Jerris R, Wang YF, Farley MM. (2012). Prevalence of blaZ Gene types and the Inoculum Effect with Cefazolin among Bloodstream Isolates of Methicillin-susceptible Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. p.4474 – 4477. Vol. 56. Number 8 Lowy F. (1998). Staphylococcus aureus infections. The New England Journal of Medicine. Vol. 339, No. 8. August 20. 520-532 Lowy F. (2003) Antimicrobial resistance: the example of Staphylococcus aureus. Journal of Clinical Investigation; 111 (9): 1265-1273. Lozano C, Torres C. (2017). Actualización en la resistencia antibiótica en Gram positivos. Enfermedades Infecciosas y Microbiología Clínica. 35(Supl 1):2-8 Luria, S. E. (1946). A test for penicillin sensitivity and resistance in Staphylococcus. Proceedings of the Society for Experimental Biology and Medicine, 61(1), 46-51 Maiden M.C. (2006). Multilocus sequence typing bacteria. Annu Rev Microbiol. 60: 561 - 88 Medina E, Goldmann O. (2018). Staphylococcus aureus strategies to evade the host acquired immune response. International Journal of Medical Microbiology. Aug;308(6): 625 – 630 Martineau F., Picard F.J., Lansac N., Ménard C., Roy P.H., Ouellette M., Bergeron M.G. (2000). Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Feb;44(2): 231 – 8. McGuinness, W.A., Malachowa N., DeLeo F. (2017). Vancomycin Resistance in Staphylococcus aureus. Yale J Biol Med. 90, pp. 269 – 281 McNeil JC, Sommer LM, Boyle M, Hogan P, Vallejo JG, Hultén KG, Flores AR, Kaplan SL, Fritz S. (2020). Cefazolin inoculum effect and methicillin-susceptible Staphylococcus aureus osteoarticular infections in children. Antimicrobial Agents and Chemotherapy 64: e00703- 20. Miller W.R., Seas C., Carvajal L.P., Diaz L., Echeverri A.M., Ferro C., Rios R., Porras P., Luna C., Gotuzzo E., Munita J.M., Nannini E., Carcamo C., Reyes J., Arias C.A. (2018). The cefazolin Inoculum Effect is Associated with increased mortality in Methicillin-Susceptible Staphylococcus aureus bacteremia. Open Forum Infectious Diseases. Volume 5, Issue 6, June, ofy123 Monson LS. (2011). “Staphylococci”. En Mahon CR., Lehman DC., Manuselis G (ed.). Textbook of Diagnostic Microbiology. 4th edition. p. 316 – 329. Maryland, Missouri. Elsevier. Munita JM., Arias C. (2016) Mechanism of Antibiotic Resistance. Microbiology Spectrum. April;4(2). doi: 10.1128/microbiolspec.VMBF-0016-2015. Nannini EC., Singh KV., Murray BE. (2003). Relapse of Type A b - lactamase – producing Staphylococcus aureus Native Valve Endocarditis during Cefazolin Therapy: Revisiting the Issue. Clinical Infectious Diseases. 2003; 37:1194 - 8 Nannini E, Stryjewski ME, Singh KV, Rude TH., Corey GR., Fowler VG Jr., Murray BE. (2009) Inoculum Effect with Cefazolin among Clinical Isolates of Methicillin-susceptible Staphylococcus aureus: Frequency and Possible Cause of Cefazolin treatment Failure. Antimicrobial Agents Chemotherapy. Aug. 53(8); 3437-41. Nightingale CH., Greene DS., Quintiliani. (1975) Pharmacokinetics and clinical use of cephalosporin antibiotics. Journal of Pharmaceutical Sciences. Dec;64(12):1899-926. doi: 10.1002/jps.2600641202 Nishida, M., Matsubara, T., Murakawa, T., Mine, Y., Yokota, Y., Goto, S., Kuwahara, S. (1970). Cefazolin, a new semisynthetic cephalosporin antibiotic. II. Journal of Antibiotics, 23(3), 137-148 Nulens, E., Stobberingh, E.E., van Dessel, H., Sebastian, S., van Tiel, F.H., Beisser, P.S., Deurenberg, R.H., (2008). Molecular characterization of Staphylococcus aureus bloodstream isolates collected in a Dutch university hospital between 1999 and 2006. Journal of Clinical Microbiology. 46, 2438–2441 O ́Riordan K, (2004). Lee JC. Staphylococcus aureus Capsular Polysaccharides. Clinical Microbiology Reviews. 17(1): 218. DOI: 10.1128/CMR.17.1.218-234. Otto M. (2012). MRSA virulence and spread. Cell Microbiology. 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Staphylococcus aureus Secreted Toxins and Extracellular Enzymes. Microbioloby Spectrum 7(2): GPP3-0039-2018 Tan L, Li SR, Jiang B, Hu XM and Li S (2018) Therapeutic Targeting of the Staphylococcus aureus Accessory Gene Regulator (agr) System. Frontiers in Microbiology. 9:55. doi: 10.3389/fmicb.2018.00055 Unnikrishnan M, Constantinidou C, Palmer T, Pallen MJ. (2017). The Enigmatic Esx Proteins: Looking Beyond Mycobacteria. Trends in Microbiology. Mar;25(3): 192 – 204 Urish KL., Cassat JE. (2020). Staphylococcus aureus osteomyelitis: bone, bugs and surgery. Infection and Immunity. Jun 22; 88(7): e00932-19. doi: 10.1128/IAI.00931-19 Vasquez MT., Lubkin A, Reyes T, Day CJ, Lacey K, Jennings MP., Torres VJ. (2020). Identification of a domain critical for Staphylococcus aureus LukED receptor targeting and lysis of erythrocytes. Journal of Biological Chemistry. Dec 11; 295(50): 17241 - 17250 Visansirikul S, Kolodziej SA, Dmenchenko AV. (2020). Staphylococcus aureus capsular polysaccharides: a structural and synthetic perspective. Organic and Biomolecular Chemistry Journal. Feb 7;18(5): 783-798. Voladri R.K., Kernodle D.S. (1998). Characterization of a chromosomal gene encoding type B b - lactamase in phage group II isolates of Staphylococcus aureus. Antimicrob Agents Chemother. Dec;42(12): 3163 – 8. Wang S., Gilchrist A., Loukitcheva A., Plotkin BJ., Sigar IM., Gross AE., O ́Donnell JN., Pettit N., Buros A., O ́Driscoll T., Rhodes NJ., Bether C., Segreti J., Charnot-Katsikas A., Singh K., Scheetz MH. (2018). Prevalence of a Cefazolin Inoculum Effect Associated with blaZ Genes Types among Methicillin-susceptible Staphylococcus aureus Isolates from Four Major Medical Centers in Chicago. Antimicrobial Agents Chemotherapy. Jul 27;62 (8). Weinstein, A. (1980). The Cephalosporins: Activity and Clinical use. Drugs 19: 137-154. Wilke M, Hills TL., Zhang HZ., Chambers HF., Strynadka CJ. (2004). Crystal Structures of the Apo and Penicillin-acylated Forms of the BlaR1 B-lactam Sensor of Staphylococcus aureus. The Journal of Biological Chemistry. Vol. 279, No. 45. Nov 5, pp. 47278 – 47287. Wong D., Wong T., Romney M., Leung V. (2016). Comparative effectiveness of b-lactam versus vancomycin empiric therapy in patients with methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. Annals of Clinical Microbiology and Antimicrobials. Apr 26; 15:27. doi: 10.1186/s12941-016-0143-3 Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. (2012). Identification of acquired antimicrobial resistance genes. Journal of Antimicrobial Chemotherapy 67:2640–2644. Zapun A., Contreras-Martel C., Vernet T. (2008) Penicillin-binding proteins and b-lactam resistance. FEMS Microbiology Reviews 32. 361-385 Zecconi A, Scali F. (2013). Staphylococcus aureus virulence factors in evasion from innate immune defenses in human and animal diseases. Immunology Letters. Feb;150(1-2): 12-22 Zhang HZ., Hackbarth CJ., Chansky KM., Chambers HF. (2001). A proteolytic transmembrane signaling pathway and resistance to beta – lactams in Staphylococci. Science. 291: 1962 – 1965. Zygmunt D., Stratton C., Kernodle D. (1992). Characterization of Four b - lactamases Produced by Staphylococcus aureus. Antimicrob. Agents Chemother. Feb;36(2): 440 – 5. |
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Atribución-NoComercial 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Reyes Manrique, Jinnethe Cristina1a7959aca115b80940d79ad4deeb38b6Reguero Reza, María Teresa Jesús9ba54bc0326cdc978d84a9a9130f2d0cSoto Guzmán, Fredi Giovannic47f053c44084feedf1eb6a440cef79bUnidad de Genética y Resistencia antimicrobiana - UGRA. Universidad El Bosque. Bogotá D.C.2023-08-08T17:21:48Z2023-08-08T17:21:48Z2022-12https://repositorio.unal.edu.co/handle/unal/84491Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/ilustraciones, diagramasStaphylococcus aureus (S. aureus) es un microorganismo versátil, que puede ser colonizador de la piel y las mucosas de los vertebrados, siendo también capaz de causar enfermedades graves en los hospederos. Aunque se describe mundialmente el aumento de los aislamientos de S. aureus resistentes a meticilina (MRSA por sus siglas en inglés), las cepas de S. aureus sensibles a meticilina (MSSA por sus siglas en inglés) continúan produciendo infecciones graves, que causan una morbimortalidad alta. La presencia de efecto inóculo a cefazolina es un fenómeno descrito desde hace aproximadamente 50 años, pero poco estudiado y comprendido. Comprobar que una infección por S. aureus meticilino sensible es causada por una cepa con presencia de este fenómeno de manera rápida, no es posible con metodología estándar y tampoco se conoce demasiado sobre los factores de resistencia y virulencia asociados en estas cepas. La presencia del efecto inóculo, puede hacer inútil el tratamiento con cefazolina cuando de forma empírica se utiliza o cuando con resultado del antibiograma, es reportada como sensible a este antibiótico, llevando a falla terapéutica. El presente estudio tiene como objetivos: i) establecer la prevalencia de efecto inóculo a cefazolina en 186 aislamientos de MSSA provenientes de hemocultivos positivos (bacteriemia) de pacientes colombianos entre los años, 2011 a 2013, ii) identificar cambios genéticos específicos asociados al efecto inóculo en el operón blaZ e iii) identificar genes de resistencia a diferentes tipos de antibióticos y genes de virulencia en estas cepas. Las MIC para Cz fueron determinadas con inóculos bacterianos estándar de 5 x 105 UFC/mL y alto inóculo de 5 x 107 UFC/mL, por microdilución en caldo BHI, que se dejaron en incubación por 24 horas a temperatura de 37ºC. Se utilizaron rangos entre 0.0625 µg/mL, hasta 64 µg/ml. Se dejaban estas placas en incubación y se realizó la lectura e interpretación adecuada. Este experimento se repitió tres veces con cada cepa y la observación de cada pozo fue realizada por tres observadores diferentes. Se consideró que existía el EICz en aquellas cepas con una concentración mayor o igual a 16 µg/mL. El control de calidad se realizó utilizando cepas de referencia como i) la cepa TX0117, la cual es productora de alta cantidad de b - lactamasa tipo A con MIC de > 64 µg/mL; ii) la cepa ATCC29213, cepa productora de pequeña cantidad de b - lactamasa tipo A con MIC de 2 a 4 µg/mL y iii) la cepa ATCC25923, cepa b - lactamasa negativa con MIC de 0.25 a 0.5 µg/mL. A estas cepas se les realizo la extracción de DNA, empleando el estuche comercial DNeasey Blood & Tissue Kit, Quiagen y la cuantificación del ADN genómico se realizó por flurometría empleando Qubit 2.0, la preparación de librerías se utilizó el estuche comercial Nextera XT (illumina); su verificación se hizo con fluorometría (Qubit 2.0) y se empleó el equipo Agilent 2100 Bioanalyzer para su normalización y la secuenciación genómica se realizó en la plataforma illumina (MiSeq). Las lecturas de secuenciación fueron procesadas eliminando posibles contaminaciones y descartando lecturas de baja calidad mediante Trimmomatic; las lecturas fueron reensambladas con SPAdes y anotadas con RAST. Las búsquedas de cada genoma se realizaron con el programa BLASTX, contra las bases de datos ResFinder y VFDB. En la determinación de la secuenciación de blaZ, para identificar diferencias específicas de los aminoácidos en los residuos 128 – 216, se realizó a partir de un lineamiento múltiple de secuencias de la proteína con MUSCLE. El resistoma y el viruloma se realizó de acuerdo a la búsqueda en los genomas de 3078 genes de resistencia y 3659 de genes de virulencia en plataformas de información como ResFinder y Virulence factors database. De los 186 aislamientos, 73 (39%) tenían presencia del efecto inóculo (EI) a cefazolina (Cz) [EICz], mientras que 113 aislamientos (61%) no lo tenían. Las b - lactamasas más asociadas a la presencia del EICz fueron la tipo A y la C y los alotipos BlaZ – 2 y BlaZ – 1 pertenecientes a los “complejos clonales (CC)” 30 y 8, respectivamente, adicional fueron los alotipos con mayor probabilidad de presentar el fenómeno estudiado. En las cepas sin EICz, se encontró que la b - lactamasa tipo B fue la más asociada a ausencia del fenotipo. Los alotipos más frecuentes fueron BlaZ – 3, BlaZ – 5 y BlaZ – 7 y los CC5, CC8 y CC1 fueron los más detectados en este grupo. En 24 de las 73 cepas con presencia del EICz, (32%) se encontró al menos un gen de resistencia a otros antibióticos como: parC para quinolonas, rpoB para rifampicina, ant(9) - Ia para aminoglicósidos, tet(K) para tetraciclinas y erm(A) para macrólidos que no son antibióticos considerados de primera línea para el tratamiento de infecciones por MSSA. En ausencia del EICz, 93 de 113 cepas (82%), se encontró al menos un gen de resistencia de los ya mencionados, junto con otros como fusB de resistencia al ácido fusídico, dfrA8 de resistencia al trimetoprim y más enzimas modificadores de aminoglicósidos como aadD, aph(3´) – III, además, el gen rpoB no fue encontrado en este grupo de aislamientos sin el fenotipo. Los genes de virulencia muestran más diferencias entre los aislamientos con y sin presencia del efecto, pero la mayoría de ellas coinciden en la ausencia de genes como los asociados a la producción de leucocidinas como PVL, enterotoxinas, toxinas exfoliativas, coagulasa entre otros. En todas las cepas, se identificó el sistema regulador de genes accesorios (agr) y su relación con presencia o ausencia de EICz. A partir de este trabajo se pudo concluir que la frecuencia del EICz en Colombia es de 39%, siendo las b – lactamasas tipo A y C y los alotipos BlaZ – 2 de la b – lactamasa tipo A y BlaZ – 1 de la b – lactamasa tipo C las más frecuentes. Se observó muy baja prevalencia de genes de resistencia a otros antibióticos diferentes de los B-láctamicos en las cepas con presencia de EICz. En cuanto a la virulencia, todas ellas presentaron gran cantidad y diversidad de genes. En cuanto a las cepas que presentaron ausencia del fenotipo correspondieron al 61% y esta se observó principalmente en aislamientos con b – lactamasa tipo B. Los alotipos BlaZ – 3, BlaZ – 5 y BlaZ – 7 fueron los más frecuentes en cepas sin EICz. Se encontraron genes de resistencia a otros antibióticos en mayor frecuencia y tenían gran cantidad y variedad de genes de virulencia en cepas sin presencia del EIC, lo cual muestra que no existe ninguna relación entre su perfil de resistencia y virulencia y la presencia del fenotipo. Finalmente, el sistema agrIII se relacionó más con la presencia de EICz, mientras que el sistema agrII se relacionó con la ausencia de EICz. (Texto tomado de la fuente)Staphylococcus aureus (S. aureus) is a versatile microorganism that colonizes the human skin and nasal mucous, as well as cause serious host infectious diseases. Although the increase of methicillin-resistant S. aureus (MRSA) isolates is reported worldwide, the methicillin-sensitive S. aureus (MSSA) continues to cause serious infections and high morbidity and mortality. The presence of cefazolin inoculum effect (CzIE) is a phenomenon described for approximately 50 years ago, but its mechanism is unknown. The presence of this phenomenon is not possible with standard methodology and neither is much known about the resistance and virulence factors associated with these strains. The presence of the CzIE may render treatment with cefazolin useless when it is used empirically or when as a result of the antibiogram is reported as sensitive leading to therapeutic failure. The aims of this study are: i) to establish the prevalence of the CzIE in 186 MSSA isolates from bloodstream cultures (bacteremia) of Colombian patients between 2011 to 2013, ii) identify specific genetic changes associated with the CzIE in blaZ operon and iii) identify resistance genes to different antibiotics groups and virulence genes in these strains. MICs for Cz were determinated with standard bacterial inoculum of 5 x 105 CFU/mL and high inoculum of 5 x 107 CFU/mL, by microdilution in BHI broth, which were left in incubation for 24 hours at a temperature of 37ºC. For all strains, 5µL were served in plates with Cz. The initial concentration of the antibiotic was 10.000µg/mL, from which dilutions were made to obtain concentrations of 0.0625 µg/mL, up to 64µg/ml. Of each antibiotic preparation, 95µL were taken. These plates were incubated for 24 hours and the appearance of a precipitate or where there was a significant turbidity of the same was observed. This experiment was repeated three times with each strain and the observation of each one was carried out by three different observers. EICz was considered to exist when a precipitate appeared at MIC ³ 16 µg/mL. These strains underwent nucleic acid extraction, libraries preparation and genomic sequencing. Three of the rows of the plates were used to collect control strains for high inoculum. The strains were TX0117: high – quantity producer type A b – lactamase with MIC > 64 µg/mL, ATCC29213: small – quantity producer type A b – lactamase with MIC is 2 to 4 µg/mL and ATCC25923, strain b – lactamase negative with MIC of 0.25 to 0.5 µg/mL. DNA extraction was performed using the commercial DNeasy Blood & Tissue Kit, Quiagen. Genomic DNA quantification was performed by fluorometry using the Qubit 2.0 Fluorometer. Genomic libraries were prepared using the commercial Nextera XT kit (illumina). The verification was done with fluorometry (Qubit 2.0) and the Agilent 2100 Bioanalyxer equipment was used for its normalization. Genomic sequencing was performed on the Illumina platform in MiSeq equipment, to obtain paired sequences of 250 nucleotides. Reading sequences were processed to remove library contamination and discard poor quality read by Trimmomatic. The readings were reassembled with SPAdes and annoted with RAST. The searches of each genome were carried out with the BLASTX program with ResFinder and VFDB databases. Sequencing of a blaZ fragment to identify aminoacidic differences at residues 128 – 216 was performed from a multiple lining of protein sequences with MUSCLE. The resistome and the virulome were performed according to the search in the genomes of 3078 resistance genes and 3659 genes of virulence from information platforms such as ResFinder and Virulence factor database. Out of the 186 isolates, 73 (39%) had the presence of the CzIE, while 113 isolates (61%) did not. The b - lactamases types more associated with the presence of the effect were types A and C and the allotypes BlaZ – 2 of b - lactamase type A and BlaZ – 1 of b - lactamase type C (belonging to clonal complex or “CC” 30 and 8, respectively), showed the highest probability of presenting the effect. In strains without the effect, it was found that b - lactamase type B were the most associated with the absence of the effect. The most frequent allotypes were BlaZ – 3, BlaZ – 5 and BlaZ – 7. The CC5, CC8 and CC1 were the most isolated in this group. Twenty - four of the 73 strains with EICz (32%), showed at least one gene for resistance to other antibiotics such as macrolides, lincosamides, aminoglycosides, tetracyclines and quinolones was found, which are not antibiotics considered first – line for the treatment of infections by MSSA. Most were found in isolates with type A b - lactamase. Some genes founded were ParC for quinolones, rpoB for rifampicin, ant(9) – Ia for aminoglycosides, tet(K) for tetracyclines and ermA for macrolides. Ninety – three of the 113 strains without EICz, (82%), showed at least one resistance gene, to mor groups of antibiotics such as macrolides, lincosamides, aminoglycosides, tetracyclines, quinolones and fusidic acid. Most were found in isolates with type B b - lactamase. With the exception of rpoB gene that wasn´t found in this group, along with those already mentioned, genes such fusidic acid resistance fusB, trimethoprim resistance dfrA8 and other aminoglycosides modifying enzymes such as aadD, aph(3´)-III were found. The virulence genes show more differences between the isolates with and without the presence of the effect, but most of them coincide in the absence of genes such as those associated with the production of leukocidins such as PVL, enterotoxins, exfoliative toxins, coagulase and others. In all strains, the accessory gene regulatory system (agr) and its relationship with the presence or absence of EICz were identified. Conclusions: The frequency of EICz in the studied strains was 39%, being the type A and C b - lactamases, the most associated. The allotypes BlaZ – 2 of the type A b - lactamase and BlaZ – 1 of type C b - lactamase were the allotypes where the presence of EICz was found mainly. The few genes for resistance to other antibiotics found in strains with presence of EICz were found in isolates with BlaZ – 2 allotype of the type A b - lactamase and BlaZ – 1 of type C b - lactamase. In addition, all of them present a large number and diversity of virulence genes. The absence of EICz was 61% and this was observed in isolates with type B b - lactamase mainly. The allotypes BlaZ – 3, BlaZ – 5 and BlaZ – 7 of type B b - lactamase were the allotypes where the absence of EICz was found more frequently. More genes of resistance to other antibiotics were found in strains without the EICz in allotypes BlaZ – 3, BlaZ – 5 and BlaZ -7. Therefore, the absence of the EICz, does not mean the absence of other mechanism of antibiotic resistance. The agrIII system was more related to the presence of EICZ, while the agrII system was related to the absence of EICz.MaestríaMagíster en Ciencias - MicrobiologíaBiología molecular de agentes infecciososxxv, 117 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Ciencias - Maestría en Ciencias - MicrobiologíaFacultad de CienciasBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá570 - Biología::576 - Genética y evoluciónStaphylococcus aureusAntibióticosantibioticsStaphylococcus aureus meticilino sensibleEfecto inóculoCefazolinaAlotipo BlaZColombiaMethicillin – sensitive Staphylococcus aureusInoculum effectCefazolinblaZ AllotypeColombiaCaracterización genómica de Staphylococcus aureus susceptibles a meticilina con efecto inóculo en ColombiaGenomic characterization of methicillin susceptible Staphylococcus aureus with inoculum effect in ColombiaTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMColombiaAlonzo F, Torres VJ. 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Expediente RGE0375.InvestigadoresORIGINAL80007928.2022.pdf80007928.2022.pdfTesis de Maestría en Microbiologíaapplication/pdf3207002https://repositorio.unal.edu.co/bitstream/unal/84491/2/80007928.2022.pdf4c7216fee52161e6304d1845b7eabc2aMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-85879https://repositorio.unal.edu.co/bitstream/unal/84491/1/license.txteb34b1cf90b7e1103fc9dfd26be24b4aMD51THUMBNAIL80007928.2022.pdf.jpg80007928.2022.pdf.jpgGenerated Thumbnailimage/jpeg4808https://repositorio.unal.edu.co/bitstream/unal/84491/3/80007928.2022.pdf.jpg9fc53dd6f6127aaa75bda6f8f7b90071MD53unal/84491oai:repositorio.unal.edu.co:unal/844912024-07-19 23:32:20.441Repositorio Institucional Universidad Nacional de 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