Efecto individual de antibióticos y en combinación con β-Cloro-l-Alanina en la expresión de factores de virulencia relacionados con la formación de biopelículas de Staphylococcus aureus resistente a meticilina de origen intrahospitalario.
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Date
2023
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Universidad de Concepción
Abstract
Staphylococcus aureus es un importante patógeno bacteriano implicado en infecciones humanas y animales. Desde su aparición, al inicio de la era antibiótica, S. aureus resistente a meticilina (SARM) ha mostrado gran capacidad de incorporar genes que codifican determinantes de resistencia a múltiples antibióticos y un gran arsenal de factores de virulencia. Además, SARM representa un gran problema debido la persistencia de infecciones asociadas a dispositivos médicos, principalmente por la capacidad de formar biopelículas, permitiéndole resistir el tratamiento antibiótico, incluso en cepas que no presentan determinantes genéticos de resistencia, lo que finalmente ocasiona el retiro de la prótesis del paciente. En el último tiempo, se han probado alternativas al tratamiento terapéutico habitual, empleando combinaciones de antibióticos usados en la práctica clínica, o bien, utilizando compuestos capaces de desagregar biopelículas. En algunos aislados de SARM se ha demostrado que el uso de algunos antibióticos puede incluso aumentar la expresión de genes implicados en la formación de biopelículas, generando fenotipos hiperadhesivos. Por esta razón, es importante conocer la capacidad de formación de biopelículas y la epidemiología de las cepas de SARM circulantes en Chile. Recientemente, β-cloro-L-alanina (β-CLA), un compuesto con actividad antibacteriana, ha mostrado desagregar biopelículas de SARM en combinación con fosfomicina. Sin embargo, se desconoce el efecto de este compuesto en combinación con otros antibióticos usados en clínica sobre la expresión de los genes que codifican factores de virulencia asociados a la formación de biopelículas. Es por esto, que en esta tesis doctoral se planteó como objetivo general caracterizar las cepas de SARM provenientes de hospitales de Chile y determinar el efecto de antibióticos utilizados en el tratamiento de SARM y la combinación de ellos con β-CLA en la formación de biopelículas como también en la expresión de genes que codifican factores de virulencia asociados con esta capacidad de cepas de SARM aisladas en hospitales de Chile. Inicialmente, se trabajó con 50 cepas categorizadas por el Instituto de salud Pública de Chile como SARM hospitalarias, aisladas entre los años 2007 y 2017, las cuales fueron caracterizadas en función del perfil de susceptibilidad a linezolid, daptomicina y vancomicina; el tipo de cassette SCCmec; y tipificación molecular mediante macrorrestricción con enzima SmaI y electroforesis de campo pulsante (PFGE). Además se confirmó el fenotipo de meticilino resistencia mediante la prueba de susceptibilidad a cefoxitina y la detección del gen mecA por reacción en cadena de la polimerasa (PCR) convencional. Posteriormente, se realizó la secuenciación del genoma completo (Illumina® MiSeq) utilizando Unicycler v0.4.8 y Spades v3.15.4 para su ensamblaje, visualización con Bandage v0.8.1 y anotación genómica mediante Prokka. Se identificaron los loci tipo multilocus (MLST), spa type, se confirmó la tipificación del tipo de SCCmec, genes de resistencia a los antimicrobianos y genes de virulencia mediante SpeciesFinder 2.0, MLSTFinder 2.0, SCCmecFinder 1.2, spaTyper 1.0, ResFinder 4.1 y VirulenceFinder 2.0, respectivamente. La capacidad de formación de biopelículas se evaluó mediante ensayos de adherencia en placas de 96 pocillos, y la formación y erradicación de biopelículas en presencia de los antibióticos linezolid, daptomicina y vancomicina solos y asociados con β-CLA fue investigada en placas de poliestireno de 6 pocillos de fondo plano, con una lámina de acero inoxidable lisa, las que posteriormente se sometieron a tinción mediante LIVE/DEAD® Baclight™ Bacterial Viability Kit y analizadas por microscopía electrónica confocal (MEC), para evaluar viabilidad de las cepas y grosor de la biopelícula tras la exposición a los compuestos antibacterianos. Se confirmó el fenotipo meticilino resistente en las 50 cepas referidas por el ISP de Chile, además de la presencia del gen mecA. Se determinó que el 76% de las cepas de SARM presentaban el SCCmec tipo I, 14% SCCmec tipo II, 8% el SCCmec tipo IVa y 1 cepa (2%) el SCCmec tipo IVc. El MLST determinó que los tipos de secuencia (ST) prevalentes fueron el ST5 (76%), seguido de los ST105 (14%), ST8 (4%) y los ST5575, ST2802, y ST72 con 2% de prevalencia. El spa type t149 fue prevalente, encontrándose en el 33% de las cepas, seguido del t002 en el 12% de éstas. En cuanto al agr predominante, grupo agr era tipo II (30%) fue el más presente, y en la misma línea, la tipificación del polisacárido capsular determinó que el tipo capsular prevalente era el cap8 (82%), seguido de cap5 (18%). Sin embargo, se descartaron 5 cepas clasificadas originalmente como SARM de origen hospitalaria, pero la tipificación molecular de los SCCmec determinó su origen comunitario. Y de acuerdo al criterio de inclusión no se siguióo trabajando con estas cepas. Las cepas presentaron un perfil de multirresistencia, destacando un 98% de resistencia a los antibióticos del grupo MLSB, sobre 98% a quinolonas y 64% a gentamicina. No se encontraron cepas resistentes a cotrimoxazol, linezolid (LZD), vancomicina (VAN) o daptomicina (DAP). Concordantemente, las cepas presentaron una alta prevalencia de genes de resistencia a distintas familias de antibióticos, como son los que codifican enzimas modificantes de aminoglucósidos: aac(6')-Ie-aph(2'')-Ia (82%), aad(6) y ant(6)-Ia (64%), aadD (16%), aadE (7%), ant(9)-Ia (98%), ant(4')-Ib (16%), sat4 (40%), aph(3')-IIIa (64%), enzimas metilasas: ermA (98%), ermC (4%), betalactamasa: blaZ (82%), rifampicina fosfotransferasa: rphB (91%), y dihidrofolato reductasa: dfrC (1%). Además, todas las cepas presentaron los genes fosB (resistencia a fosfomicina), norA, mgrA, mepA, mepR, sav1866 (resistencia a fluoroquinolonas), tet38 (resistencia a tetraciclinas) y el sistema regulador de 2 componentes arlRS. Respecto a la caracterización de genes que codifican factores de virulencia implicados en la formación de biopelículas, el 98% de las cepas presentaron el gen fnbA y 26% el gen fnbB (codifiantres de las proteínas de unión a fibronectina A y B, respectivamente); 98% el operón icaABCDR; 80% hly/hla y el todas portaban los genes hlb y hld, codificantes de las hemolisinas b y d, respectivamente. No se detectó la presencia del gen bap, ni de los operones pmsA y pmsB en ninguna de las 45 cepas ensayadas. Sobre el 85% de las cepas presentaron otros genes de virulencia asociados a la adhesión, producción de exoenzimas, toxinas, enterotoxinas y la captación de hierro. Los estudios de relación genética mostraron la presencia de 5 clados, destacando el clado principal con cepas portadoras del SCCmec I distribuídas en la zona centro sur de Chile entre los años 2012 al 2017. Los estudios de formación de biopelículas indicaron que sólo el 73% de las cepas eran productoras de biopelículas, y que el 55% y el 18% del total mostraron un índice de formación de biopelículas débil (DFBP) y moderado (MFBP), respectivamente. De estas cepas se seleccionó una de cada fenotipo y se realizaron ensayos de formación y erradicación de biopelículas en presencia de VAN, DAP, LZD y β-CLA separadamente, y en en combinación con este último. Los análisis de MEC indicaron que la cepa no formadora de biopelículas presentó un aumento de la biomasa viva de la biopelícula madura en concentraciones sub-CMI de VAN, DAP y β-CLA, y una disminución frente a LZD. Las cepas con fenotipo NFBP y MFBP mostraron un aumento en la lectura del espectro rojo cuando se expusieron a concentraciones sub-CMI de LZD, y de las combinaciones LZD + β-CLA, VAN + β-CLA y DAP + β-CLA (p= 0,019) y sub-CMI de VAN, DAP, β-CLA, LZD + β-CLA, VAN + β-CLA y DAP+ β-CLA (p=0,037), respectivamente; lo cual indica que a estas concentraciones ensayadas se induce la muerte celular dentro en la biopelícula, permitiendo además su desagregación.
Las cepa con fenotipo DFBP no mostró cambios en la producción de biopelículas frente a los compuestos ensayados. Los ensayos de expresión de genes de virulencia no arrojaron resultados concluyentes con la metodología ensayada. Las cepas de SARM-AH referidas por el ISP de Chile analizadas en esta tesis, presentan prevalentemente, a nivel tanto fenotípico como genotípico, el perfil clásico del clon chileno/cordobés con un amplio arsenal de genes virulencia, principalmente codificantes de factores de adherencia, exoenzimas, toxinas y hemolisinas. Por otra parte, el uso de sub-CMI β-CLA, VAN y DAP contribuyen al a formación de biopelículas en una cepa con fenotipo no productor de biopelículas. El uso de combinaciones a concentraciones sub-CMI de 1 μg/mL LZD, y de las combinaciones de 1 μg/mL LZD +16 μg/mL β-CLA, 0,5 μg/mL VAN + 16 μg/mL β-CLA y 0,125 μg/mL de DAP + 16 μg/mL β-CLA, provocan una disminución de la formación de esta biopelícula, favoreciendo su erradicación.
Staphylococcus aureus is an important bacterial pathogen implicated in human and animal infections. ince the dawn of the antibiotic era, methicillin-resistant Staphylococcus aureus (MRSA) has demonstrated a remarkable ability to acquire genes responsible for resistance to multiple antibiotics, along with a vast array of virulence factors.. In addition, MRSA represents a great problem due to the persistence of infections associated with medical devices, mainly due to the ability to form biofilms, allowing it to resist antibiotic treatment, even in strains that do not present genetic determinants of resistance, which ultimately necessitates the removal of the patient's drug-impregnated medical device. In recent times, alternatives to the usual therapeutic treatment have been tested, using combinations of antibiotics employed in clinical practice and compounds capable of disaggregating biofilms. In some MRSA isolates, it has been shown that the use of some antibiotics can even increase the expression of genes involved in the formation of biofilms, generating hyperadhesive phenotypes. For this reason, it is important to know the biofilm formation capacity and the epidemiology of circulating MRSA strains in Chile. Recently, β-chloro-L-alanine (β-CLA), a compound with antibacterial activity, has been shown to disaggregate MRSA biofilms in combination with fosfomycin. However, the effect of this compound in combination with other clinically used antibiotics on the expression of genes encoding virulence factors associated with biofilm formation is unknown. For this reason, in this doctoral thesis, the general objective was to characterize the MRSA strains from hospitals in Chile and to determine the effect of antibiotics used in the treatment of MRSA and the combination of them with β-CLA on the formation of biofilms and also to determine the impact on gene expression for virulence factors associated with this biofilm-forming ability in MRSA strains isolated from Chilean hospitals. We worked with 50 hospital MRSA strains provided by the Chilean Public Health Institute, isolated between 2007 and 2017, which were characterized based on the susceptibility profile to linezolid, daptomycin, and vancomycin; the SCCmec cassette type; and molecular typing by macrorestriction with SmaI enzyme and pulsed field electrophoresis (PFGE). In addition, the methicillin resistance phenotype was confirmed by means of the cefoxitin susceptibility test and the detection of the mecA gene by conventional polymerase chain reaction (PCR). Subsequently, whole genome sequencing (Illumina® MiSeq) was performed using Unicycler v0.4.8 and Spades v3.15.4 for assembly, visualization with Bandage v0.8.1, and genomic annotation using Prokka. Multilocus type loci (MLST), spa type, SCCmec typing, antimicrobial resistance genes, and virulence genes were confirmed using SpeciesFinder 2.0, MLSTFinder 2.0, SCCmecFinder 1.2, spaTyper 1.0, ResFinder 4.1, and VirulenceFinder 2.0., respectively. Biofilm formation capacity was assessed by adhesion assays in 96-well plates, and biofilm formation and eradication in the presence of the antibiotics linezolid, daptomycin and vancomycin alone and associated with β-CLA was investigated in 6-well polystyrene plates flat bottom wells, with a smooth stainless steel sheet, which were subsequently stained using the LIVE/DEAD® Baclight™ Bacterial Viability Kit and analyzed by confocal electron microscopy (CEM), to assess viability of the strains and thickness of the biofilm after exposure to antibacterial compounds. The methicillin-resistant phenotype, along with the presence of the mecA gene, was confirmed in all 50 strains reported by the Chilean Public Health Institute (ISP), in addition to the presence of the mecA gene. It was determined that 76% of the MRSA strains had SCCmec type I, 14% SCCmec type II, 8% SCCmec type Iva, and 1 strain (2%) SCCmec type Ivc. The MLST determined that the prevalent sequence types (ST) were ST5 (76%), followed by ST105 (14%), ST8 (4%), and ST5575, ST2802, and ST72 with 2% prevalence. The spa type t149 was the most prevalent, identified in 33% of the strains, with t002 found in 12%. Regarding the predominant agr, agr group was type II (30%) was the most present, and in the same line, the typing of the capsular polysaccharide determined that the prevalent capsular type was cap8 (82%), followed by cap5 (18 %). However, molecular typing of SCCmec revealed that 5 strains initially classified as hospital-associated MRSA were actually of community origin. In accordance with the inclusion criteria, these strains were excluded from further study. The strains showed a multiresistance profile, highlighting 98% resistance to antibiotics of the MLSB group, more than 98% to quinolones, and 64% to gentamicin. No strains resistant to cotrimoxazole, linezolid (LZD), vancomycin (VAN) or daptomycin (DAP) were found. Concordantly, the strains presented a high prevalence of resistance genes to different families of antibiotics, such as those encoding aminoglycoside-modifying enzymes: aac(6')-Ie-aph(2'')-Ia (82%), aad(6) and ant(6)-Ia (64%), aadD (16%), aadE (7%), ant(9)-Ia (98%), ant(4')-Ib (16%), sat4 (40%), aph(3')-IIIa (64%), methylase enzymes: ermA (98%), ermC (4%), beta-lactamase: blaZ (82%), rifampin phosphotransferase: rphB (91%), and dihydrofolate reductase: dfrC (1%). In addition, all the strains presented the genes fosB (resistance to fosfomycin), norA, mgrA, mepA, mepR, sav1866 (resistance to fluoroquinolones), tet38 (resistance to tetracyclines) and the 2-component regulatory system arlRS. Regarding the characterization of genes that encode virulence factors involved in the formation of biofilms, 98% of the strains presented the fnbA gene and 26% the fnbB gene (encoding fibronectin binding proteins A and B, respectively); 98% the icaABCDR operon; 80% hly/hla and all carried the hlb and hld genes, encoding hemolysins b and d, respectively. The presence of bap gene, or pmsA and pmsB operons was not detected in any of the 45 strains tested. Over 85% of the strains presented other virulence genes associated with adhesion, production of exoenzymes, toxins, enterotoxins, and iron uptake. The genetic relationship studies showed the presence of 5 clades, highlighting the main clade with strains carrying SCCmec I distributed in the south-central zone of Chile between 2012 and 2017. The biofilm formation studies indicated that only 73% of the strains were biofilm producers, and that 55% and 18% of the total showed a weak (DFBP) and moderate (MFBP) index of biofilm formation, respectively. One of each phenotype was selected from these strains and biofilm formation and eradication assays were performed in the presence of VAN, DAP, LZD and β-CLA separately, and in combination with the latter. The MEC analyzes indicated that the non-biofilm-forming strain showed an increase in live biomass of the mature biofilm at sub-MIC concentrations of VAN, DAP and β-CLA, and a decrease compared to LZD. Strains with NFBP and MFBP phenotypes showed an increase in the red spectrum reading when exposed to sub-CMI concentrations of LZD, and of the combinations LZD + β-CLA, VAN + β-CLA and DAP + β-CLA (p = 0.019) and sub-MIC of VAN, DAP, β-CLA, LZD + β-CLA, VAN + β-CLA and DAP+ β-CLA (p=0.037), respectively; which indicates that at these tested concentrations cell death is induced within the biofilm, also allowing its disaggregation.Strains with DFBP phenotype did not show changes in biofilm production against the tested compounds. Virulence gene expression assays did not yield conclusive results with the tested methodology. The MRSA-HA strains referred to by the ISP of Chile analyzed in this thesis present predominantly, at both the phenotypic and genotypic level, the classic profile of the Chilean/Cordobes clone with a wide arsenal of virulence genes, mainly encoding adhesion factors, exoenzymes, toxins and hemolysins. On the other hand, the use of sub-CMI β-CLA, VAN and DAP contribute to the formation of biofilms in a strain with a non-biofilm-producing phenotype. The use of combinations at sub-MIC concentrations of 1 μg/mL LZD, and of combinations of 1 μg/mL LZD +16 μg/mL β-CLA, 0.5 μg/mL NPV + 16 μg/mL β-CLA and 0.125 μg/mL of DAP + 16 μg/mL β-CLA, cause a decrease in the formation of this biofilm, favoring its eradication.
Staphylococcus aureus is an important bacterial pathogen implicated in human and animal infections. ince the dawn of the antibiotic era, methicillin-resistant Staphylococcus aureus (MRSA) has demonstrated a remarkable ability to acquire genes responsible for resistance to multiple antibiotics, along with a vast array of virulence factors.. In addition, MRSA represents a great problem due to the persistence of infections associated with medical devices, mainly due to the ability to form biofilms, allowing it to resist antibiotic treatment, even in strains that do not present genetic determinants of resistance, which ultimately necessitates the removal of the patient's drug-impregnated medical device. In recent times, alternatives to the usual therapeutic treatment have been tested, using combinations of antibiotics employed in clinical practice and compounds capable of disaggregating biofilms. In some MRSA isolates, it has been shown that the use of some antibiotics can even increase the expression of genes involved in the formation of biofilms, generating hyperadhesive phenotypes. For this reason, it is important to know the biofilm formation capacity and the epidemiology of circulating MRSA strains in Chile. Recently, β-chloro-L-alanine (β-CLA), a compound with antibacterial activity, has been shown to disaggregate MRSA biofilms in combination with fosfomycin. However, the effect of this compound in combination with other clinically used antibiotics on the expression of genes encoding virulence factors associated with biofilm formation is unknown. For this reason, in this doctoral thesis, the general objective was to characterize the MRSA strains from hospitals in Chile and to determine the effect of antibiotics used in the treatment of MRSA and the combination of them with β-CLA on the formation of biofilms and also to determine the impact on gene expression for virulence factors associated with this biofilm-forming ability in MRSA strains isolated from Chilean hospitals. We worked with 50 hospital MRSA strains provided by the Chilean Public Health Institute, isolated between 2007 and 2017, which were characterized based on the susceptibility profile to linezolid, daptomycin, and vancomycin; the SCCmec cassette type; and molecular typing by macrorestriction with SmaI enzyme and pulsed field electrophoresis (PFGE). In addition, the methicillin resistance phenotype was confirmed by means of the cefoxitin susceptibility test and the detection of the mecA gene by conventional polymerase chain reaction (PCR). Subsequently, whole genome sequencing (Illumina® MiSeq) was performed using Unicycler v0.4.8 and Spades v3.15.4 for assembly, visualization with Bandage v0.8.1, and genomic annotation using Prokka. Multilocus type loci (MLST), spa type, SCCmec typing, antimicrobial resistance genes, and virulence genes were confirmed using SpeciesFinder 2.0, MLSTFinder 2.0, SCCmecFinder 1.2, spaTyper 1.0, ResFinder 4.1, and VirulenceFinder 2.0., respectively. Biofilm formation capacity was assessed by adhesion assays in 96-well plates, and biofilm formation and eradication in the presence of the antibiotics linezolid, daptomycin and vancomycin alone and associated with β-CLA was investigated in 6-well polystyrene plates flat bottom wells, with a smooth stainless steel sheet, which were subsequently stained using the LIVE/DEAD® Baclight™ Bacterial Viability Kit and analyzed by confocal electron microscopy (CEM), to assess viability of the strains and thickness of the biofilm after exposure to antibacterial compounds. The methicillin-resistant phenotype, along with the presence of the mecA gene, was confirmed in all 50 strains reported by the Chilean Public Health Institute (ISP), in addition to the presence of the mecA gene. It was determined that 76% of the MRSA strains had SCCmec type I, 14% SCCmec type II, 8% SCCmec type Iva, and 1 strain (2%) SCCmec type Ivc. The MLST determined that the prevalent sequence types (ST) were ST5 (76%), followed by ST105 (14%), ST8 (4%), and ST5575, ST2802, and ST72 with 2% prevalence. The spa type t149 was the most prevalent, identified in 33% of the strains, with t002 found in 12%. Regarding the predominant agr, agr group was type II (30%) was the most present, and in the same line, the typing of the capsular polysaccharide determined that the prevalent capsular type was cap8 (82%), followed by cap5 (18 %). However, molecular typing of SCCmec revealed that 5 strains initially classified as hospital-associated MRSA were actually of community origin. In accordance with the inclusion criteria, these strains were excluded from further study. The strains showed a multiresistance profile, highlighting 98% resistance to antibiotics of the MLSB group, more than 98% to quinolones, and 64% to gentamicin. No strains resistant to cotrimoxazole, linezolid (LZD), vancomycin (VAN) or daptomycin (DAP) were found. Concordantly, the strains presented a high prevalence of resistance genes to different families of antibiotics, such as those encoding aminoglycoside-modifying enzymes: aac(6')-Ie-aph(2'')-Ia (82%), aad(6) and ant(6)-Ia (64%), aadD (16%), aadE (7%), ant(9)-Ia (98%), ant(4')-Ib (16%), sat4 (40%), aph(3')-IIIa (64%), methylase enzymes: ermA (98%), ermC (4%), beta-lactamase: blaZ (82%), rifampin phosphotransferase: rphB (91%), and dihydrofolate reductase: dfrC (1%). In addition, all the strains presented the genes fosB (resistance to fosfomycin), norA, mgrA, mepA, mepR, sav1866 (resistance to fluoroquinolones), tet38 (resistance to tetracyclines) and the 2-component regulatory system arlRS. Regarding the characterization of genes that encode virulence factors involved in the formation of biofilms, 98% of the strains presented the fnbA gene and 26% the fnbB gene (encoding fibronectin binding proteins A and B, respectively); 98% the icaABCDR operon; 80% hly/hla and all carried the hlb and hld genes, encoding hemolysins b and d, respectively. The presence of bap gene, or pmsA and pmsB operons was not detected in any of the 45 strains tested. Over 85% of the strains presented other virulence genes associated with adhesion, production of exoenzymes, toxins, enterotoxins, and iron uptake. The genetic relationship studies showed the presence of 5 clades, highlighting the main clade with strains carrying SCCmec I distributed in the south-central zone of Chile between 2012 and 2017. The biofilm formation studies indicated that only 73% of the strains were biofilm producers, and that 55% and 18% of the total showed a weak (DFBP) and moderate (MFBP) index of biofilm formation, respectively. One of each phenotype was selected from these strains and biofilm formation and eradication assays were performed in the presence of VAN, DAP, LZD and β-CLA separately, and in combination with the latter. The MEC analyzes indicated that the non-biofilm-forming strain showed an increase in live biomass of the mature biofilm at sub-MIC concentrations of VAN, DAP and β-CLA, and a decrease compared to LZD. Strains with NFBP and MFBP phenotypes showed an increase in the red spectrum reading when exposed to sub-CMI concentrations of LZD, and of the combinations LZD + β-CLA, VAN + β-CLA and DAP + β-CLA (p = 0.019) and sub-MIC of VAN, DAP, β-CLA, LZD + β-CLA, VAN + β-CLA and DAP+ β-CLA (p=0.037), respectively; which indicates that at these tested concentrations cell death is induced within the biofilm, also allowing its disaggregation.Strains with DFBP phenotype did not show changes in biofilm production against the tested compounds. Virulence gene expression assays did not yield conclusive results with the tested methodology. The MRSA-HA strains referred to by the ISP of Chile analyzed in this thesis present predominantly, at both the phenotypic and genotypic level, the classic profile of the Chilean/Cordobes clone with a wide arsenal of virulence genes, mainly encoding adhesion factors, exoenzymes, toxins and hemolysins. On the other hand, the use of sub-CMI β-CLA, VAN and DAP contribute to the formation of biofilms in a strain with a non-biofilm-producing phenotype. The use of combinations at sub-MIC concentrations of 1 μg/mL LZD, and of combinations of 1 μg/mL LZD +16 μg/mL β-CLA, 0.5 μg/mL NPV + 16 μg/mL β-CLA and 0.125 μg/mL of DAP + 16 μg/mL β-CLA, cause a decrease in the formation of this biofilm, favoring its eradication.
Description
Tesis presentada para optar al grado de Doctor en Ciencias Mención Microbiología.