Estudio de cepas de Corynebacterium pseudotuberculosis, agente etiológico de Linfoadenitis Caseosa, en pequeños rumiantes.
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2020
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Universidad Nacional de la Patagonia San Juan Bosco. Facultad de Ciencias Naturales y Ciencias de la Salud. Departamento de Bioquímica.
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Resumen: Corynebacterium pseudotuberculosis, agente etiológico de linfoadenitis caseosa (LC), es un bacilo Gram positivo, pleomórfico con forma de cocobacilo y agrupación en empalizada.
Es inmóvil, no esporulado, no capsulado, anaerobio facultativo y desarrolla en medios de cultivos enriquecidos a 37 ºC y pH óptimo 7 a 7,2. La LC es una enfermedad infecto-contagiosa de evolución crónica, que afecta principalmente ovejas, cabras y otros animales domésticos.
En equinos y vacunos se manifiesta como linfangitis ulcerativa y en vacas lecheras puede causar mastitis. La LC en ovejas adultas se presenta en forma cutánea y/o visceral. Es considerada una zoonosis ocupacional a nivel mundial. Existen investigaciones y publicaciones sobre la enfermedad tanto en nuestro país como en Canadá, Australia, Francia, Holanda, Nueva Zelanda, Estados Unidos, Brasil, Suiza, Uruguay, Venezuela, Turquía y Cuba entre otros. Los objetivos de esta tesis fueron estudiar las características morfológicas, fisiológicas, metabólicas y de virulencia en aislamientos de C. pseudotuberculosis, de distintas localizaciones geográficas, aislados de lesiones de L C en pequeños rumiantes y determinar si existe relación entre estos aislamientos locales y de otras regiones con alta prevalencia de LC.
Se estudiaron 35 aislamientos de C. pseudotuberculosis de distintos orígenes: 7 de caprinos y 28 de ovinos, aislados de distintas lesiones y de diferentes lugares geográficos; 21 aislamientos de Rio Negro, Chubut, Santa Cruz y Tierra del Fuego (Argentina) y 14 aislamientos de los estados de San Pablo: Aracatuba y de Salvador de Bahía (Brasil). Los mismos fueron identificados y confirmados por métodos bioquímicos y moleculares. Por otro lado se determinó la sensibilidad a antibióticos por los métodos de difusión en agar y dilución.
Además, se investigó la presencia de integrones que contienen genes de resistencia. Asimismo se estudiaron factores de virulencia, entre ellos: ácidos micólicos, que se determinó mediante cromatografía en capa delgada (TLC) de los ésteres metílicos de ácidos micólicos extraídos de aislamientos de C. pseudotuberculosis; exotoxina (foslipasa D) que es el principal factor de virulencia de este microorganismo y se determinó su acción mediante las pruebas de CAMP y CAMP reversa y mediante la técnica de SDS-PAGE se obtuvo su masa molecular aparente; producción de otras enzimas extracelulares y capacidad de formación de biopelículas, para la cual se utilizó un método colorimétrico con cristal violeta al 0,1 % que permitió categorizar dicha capacidad. Mediante la técnica de microscopia electrónica de barrido, se determinó la interacción de una selección de aislamientos de C. pseudotuberculosis en líneas celulares MDBK (Madin Darb Bovine Kidney) y posteriormente efectos citopáticos sobre las células, utilizando técnicas de coloración de Giemsa y microscopia óptica. Finalmente se aplicaron sistemas de información geográfica (SIG), como herramienta de análisis espacial de aislamientos de C. pseudotuberculosis biovariedad ovis provenientes de distintos sitios geográficos con el fin de encontrar una relación entre los mismos. Los resultados obtenidos mostraron que los aislamientos fueron positivos para la producción de catalasa y ureasa; mostraron reacción variable para -glucosidasa y fosfatasa alcalina y el 100 % fueron negativos para nitratoreductasa, pirazinamidasa, pirrolidonil arilamidasa, -glucuronidasa, - galactosidasa, N-acetil--glucosaminidasa, hidrólisis de esculina y gelatina a 22 °C y a 37 °C.
Se determinó que el 100 % de los aislamientos provenientes de la Patagonia Argentina y la región noreste de Brasil, correspondían a la biovariedad ovis. Se registraron biotipos en los aislamientos en relación a la fermentación de los azúcares glucosa, galactosa y maltosa, determinándose que el 33 % de los aislamientos correspondieron al biotipo Glu+Gal-Mal- y el 67 % a Glu+Gal-Mal+. El 33 % de los aislamientos de origen ovino y provenientes de Argentina presento el biotipo Glu+Gal-Mal-. Los restantes aislamientos de Argentina al igual que los procedentes de Brasil fermentaron la maltosa, haciendo referencia al biotipo Glu+Gal-Mal+. Este biotipo se observó en un 70 % en la presentación cutánea, mientras que el biotipo Glu+Gal-Mal- se manifestó en un 60 % en la presentación visceral. Por el método de difusión, amoxicilina-clavulánico, ampicilina, cefotaxima, cefoxitina, ciprofloxacina, cloranfenicol, eritromicina, estreptomicina, gentamicina, imipenem, kanamicina, norfloxacina, penicilina, rifampicina, tetraciclina, trimetroprima–sulfametoxazol y vancomicina fueron activos frente al 100 % de los aislamientos, mientras que amicacina y oxacilina presentaron resultados variables. En los aislamientos que desarrollaron frente a amicacina y oxacilina, se investigó la presencia de integrones de clase 1. El resultado fue negativo, sugiriendo la ausencia del integrón asociado a la resistencia antibiótica. Utilizando el método de dilución, los antibióticos más activos correspondieron a los grupos de cefalosporinas, glucopéptidos, macrólidos, quinolonas y tetraciclinas. Se demostró menor actividad de β-lactámicos y aminoglucósidos. No se registró variabilidad en los perfiles antibióticos en los aislamientos procedentes de diferentes fuentes.
De acuerdo a los frentes de corrida (Rf) obtenidos, C. pseudotuberculosis biovariedad ovis presentó los ácidos micólicos AM tipo IV, V y VI. Los aislamientos de Argentina y de Brasil mostraron el mismo perfil de ácidos micólicos. Las pruebas de CAMP y CAMP reversa resultaron positivas poniendo en evidencia la acción de la exotoxina. La masa molecular aparente de la misma se estimó en 29,55 kDa. Se registró un 100 % de actividad hemolítica en sangre de oveja, confirmando la producción de fosfolipasa D. El 3 % de los aislamientos no presentaron hemólisis sobre eritrocitos humanos y ninguno tuvo acción sobre eritrocitos de equino, este resultado podría deberse a la composición fosfolípidica especifica de la membrana eritrocitaria de cada especie de mamífero.
Los aislamientos exhibieron el siguiente perfil enzimático: el 100 % mostró positiva la producción de catalasa, ureasa, carboxilesterasa, amilasa, hemolisina en sangre de carnero y actividad proteolítica. El 48 % y el 97 % de los aislamientos exhibieron positiva la producción de caseinasa y hemolisina en sangre humana, respectivamente. El 100 % de los aislamientos mostró negativa la producción de lecitinasa, lipasa verdadera, fenilalanina deaminasa, hemolisina en sangre de caballo y gelatinasa a 4 °C y 37 °C.
Los resultados de los diferentes ensayos confirmaron la producción de la fosfolipasa y otras enzimas y sugieren el papel de estos factores de virulencia sobre los efectos citopáticos que se observaron en los cultivos celulares durante un período de 72 h. Se demostró la capacidad de adherencia a las 6, 24 y 72 h post infección; y se observó que las bacterias provocaron los siguientes efectos citopáticos sobre las células MDBK: lisis celular, vacuolas citoplasmáticas y alteración de la relación núcleo/citoplasma. Los resultados de lisis celular fueron analizados estadísticamente. No se encontraron diferencias significativas para las 6 h post infección del cultivo, mientras que si existieron diferencias significativas a partir de las 24 h, observándose 100 % de lisis celular en el 50 % de los aislamientos estudiados. Respecto de la vacuolización citoplasmática el análisis estadístico en este caso reveló diferencias significativas desde las 6 h de iniciada la infección. El 50 % de los aislamientos provocó la vacuolización citoplasmática en más del 60 % de la monocapa celular a las 72 h postinfección.
Para el análisis de la alteración en la relación núcleo/citoplasma se observaron diferencias significativas entre los distintos aislamientos a diferentes tiempos de infección. El 50 % provocaron la alteración en la relación núcleo/citoplasma en más del 40 % de la monocapa celular a las 24 h postinfección.
Todos los aislamientos de C. pseudotuberculosis mostraron la capacidad de producir biopelículas y el mayor porcentaje correspondió a productores moderados (47 %). Entre los aislamientos de Argentina predominaron tanto los productores débiles (36 %) como los moderados (36 %) mientras que en Brasil el 75 % de los aislamientos mostraron capacidad de producción de biopelículas moderada y no se detectaron productores fuertes. La aplicación de SIG permitió el abordaje de los resultados desde una visión interdisciplinaria. La distribución dispersa de los aislamientos condujo a realizar el análisis de los mismos considerando la variable clima. Este estudio, permitió encontrar una relación entre los aislamientos provenientes de provincias de la Patagonia Argentina y estados de Brasil. Surgieron dos grupos bien diferenciados, el 74 % tuvo una relación directa con el clima seco por otra parte el 26 % de los aislamientos se ubicó en zonas asociadas a la clasificación de clima lluvioso y templado A partir de estos resultados se concluye que:
-Existe una amplia variabilidad fenotípica entre los aislamientos de C. pseudotuberculosis y presentan capacidades diferenciales en su metabolismo, sensibilidad antibiótica, grados de intensidad del daño celular y de formación de biopelículas. Conocer estas características permitió establecer aspectos diferenciales y hallar una vinculación climática-estacional con respecto a su presentación endémica en las regiones de procedencia de los aislamientos investigados.
-Los aislamientos de C. pseudotuberculosis provenientes de ambos países corresponden a la biovariedad ovis. Entre los aislamientos de Argentina y Brasil existen variantes bioquímicas para la producción de enzimas α-glucosidasa y fostafatasa alcalina y en la fermentación de la maltosa. Se destacan 2 biotipos, Glu+ Gal- Malt- y Glu+ Gal- Malt+. Siendo este último más frecuente en la presentación cutánea y el primero en ambas formas, cutánea y visceral.
-El 100 % de los aislamientos corresponden a C. pseudotuberculosis biovariedad ovis confirmados mediante la secuenciación del gen que codifica para el 16S ARNr, con un muy alto grado de similitud frente a cepas de referencia depositadas en bancos genéticos.
-Una amplia gama de antibióticos utilizados en salud pública y/o veterinaria, ensayados por el método de difusión en agar, in vitro, resultan eficaces frente a C. pseudotuberculosis biovariedad ovis. Existen entre los aislamientos resultados variables frente a amicacina y oxacilina. Los aislamientos con y sin desarrollo frente a amicacina provienen tanto de la región patagónica Argentina como de los estados de Brasil.
-En este estudio, no se detectaron integrones clase 1 en los aislamientos de C.
pseudotuberculosis biovariedad ovis que desarrollaron frente a aminoglucósidos; por lo tanto es posible que el mecanismo de resistencia este dado por otros elementos genéticos.
-En el método de dilución (CIM) in vitro, se demuestra una alta sensibilidad de C.
pseudotuberculosis biovariedad ovis frente a diferentes grupos de antibióticos, pero también menor actividad de β-lactámicos y aminoglucósidos.
-Los aislamientos de C. pseudotuberculosis biovariedad ovis procedentes de diferentes fuentes no registran variabilidad en los perfiles antibióticos.
-A pesar de las diferencias de localización geográfica, sitio de lesión y especie animal, los aislamientos de C. pseudotuberculosis biovariedad ovis de Argentina y de Brasil muestran el mismo perfil de ácidos micólicos: tipo IV, V y VI, que corresponden a cetomicolatos, epoximicolatos y micolatos dicarboxilicos, respectivamente.
-El 100 % de los aislamientos de C. pseudotuberculosis biovariedad ovis presentan la fosfolipasa D, confirmada por técnica de PCR (gen pld) y revelada en el laboratorio a través de las pruebas bioquímicas de CAMP y CAMP reversa. Ambas pruebas fueron claves para la identificación de C. pseudotuberculosis, dado que permitieron la expresión del principal factor de virulencia de este microorganismo.
-C. pseudotuberculosis biovariedad ovis es capaz de adherirse e invadir líneas celulares no fagocíticas, permaneciendo viable hasta 72 h, induciendo diferentes grados de intensidad en los efectos citopáticos estudiados.
-Los aislamientos de C. pseudotuberculosis biovar ovis, estudiados en este trabajo, demuestran virulencia debido a la producción de algunas enzimas (catalasa, caseínasa, carboxilesterasa, amilasa, ureasa, hemolisina), capacidad de adherencia e internalización en líneas celulares no fagocíticas con producción de ECP y formación de biopelículas.
-El análisis a través de SIG resulta útil para identificar como variable al clima permitiendo establecer un vínculo climático-estacional entre aislamientos de C. pseudotuberculosis biovar ovis provenientes de zonas endémicas de LC de Argentina y de Brasil.
Corynebacterium pseudotuberculosis, an etiological agent of caseous lymphadenitis (CL), is a gram-positive bacillus, pleomorphic in a coccobacillus shaped and in a palisade grouping. It is immobile, non-sporulating, non-encapsulated, facultatively anaerobic and it develops in environment enriched cultures at 37 ºC and the optimum pH at 7 to 7.2. CL is an infectious-contagious disease of chronic evolution, which mainly affects sheep, goats and other domestic animals. In horses and cows, it manifests itself as ulcerative lymphangitis and in dairy cows it can result in mastitis. CL in adult sheep can appear in cutaneous and/or visceral form. It is considered a zoonotic occupational disease worldwide. There are research studies and publications about the disease in our country as well as in Canada, Australia, France, Holland, New Zealand, United States, Brazil, Switzerland, Uruguay, Venezuela, Turkey and Cuba, among others. The objectives of this thesis were to study the morphological, physiological, metabolic and virulence factors in isolates of C. pseudotuberculosis, from various geographical areas, isolates of CL lesions in small ruminants and to determine if there is a relationship between these local isolates and other regions with high prevalence of CL. On the one hand, there were studied 35 of isolates C. pseudotuberculosis from different origins: 7 goats and 28 from sheep with different lesions and from different geographical locations. 21 separate cases from Rio Negro, Chubut, Santa Cruz and Tierra del Fuego (Argentina) and 14 isolates from the states of Sao Paulo: Aracatuba and Salvador de Bahia (Brazil). These were identified and confirmed by biochemical and molecular methods. On the other hand, the sensitivity to antibiotics was determined by agar diffusion and dilution methods. In addition, it was studied the presence of integrons containing resistance genes. Virulence factors were also studied, among them: mycolic acids, which were determined by thin-layer chromatography (TLC) of the methyl esters from mycolic acids extracted from C. pseudotuberculosis isolates. Another factor studied was exotoxin (Phospholipase D), which is the main virulence factor of this microorganism and it was determined its action through CAMP and reverse CAMP tests and through SDS-PAGE technique its apparent molecular mass was obtained. Third, the production of other extracellular enzymes and biofilm formation capacity, for which it was used a colorimetric method with violet crystal at 0.1% that allowed to classify its capacity. Through scanning electron microscopy, it was determined the interaction of a selection of C. pseudotuberculosis isolates in MDBK (Madin-Darby Bovine Kidney) cell lines and later, cytopathic effects on the cells, using Giemsa staining and optical microscopy techniques. Finally, geographic information systems (GIS) were applied as a tool for spatial analysis of C. 7 pseudotuberculosis biovari ovis isolates from different geographical sites in order to find a relationship between them. The results obtained showed that the isolates were positive for the production of catalase and urease, they also showed variable reaction for −glucosidase and alkaline phosphatase and 100 % were negative for nitrato reductase, pyrazinamidase, pyrolidonyl arylamidase, -glucuronidase, -galactosidase, N-acetyl -glucosaminidase, esculin hydrolysis and gelatin at 22 °C and 37 °C. It was determined that 100 % of the isolates from the Argentinian Patagonia and the northeast region of Brazil corresponded to the biovar ovis. There were registered biotypes in the isolates in relation to the fermentation of glucose, galactose and maltose sugars, the results determined that 33 % of the isolates corresponded to Glu+Gal-Mal- biotype and 67 % to Glu+Gal-Mal+. The 33 % of the isolates of ovine origin coming from Argentina presented the biotype Glu+Gal-Mal-. The rest of the Argentinean isolates as well as those from Brazil fermented the maltose, referring to the Glu+Gal-Mal+ biotype. This biotype was observed in 70 % of the cutaneous sample, while the biotype Glu+Gal-Mal- manifested itself in 60 % in the visceral sample. By the diffusion method, amoxicillin-clavulanic, ampicillin, cefotaxime, cefoxitin, ciprofloxacin, chloramphenicol, erythromycin, streptomycin, gentamicin, imipenem, kanamycin, norfloxacin, penicillin, rifampicin, tetracycline, trimethoprim-sulfamethoxazole and vancomycin were active in respect to 100 % of the isolates, while amikacin and oxacillin showed variable results. In the isolations that developed with amikacin and oxacillin, it was found the presence of class 1 integrons. The result was negative, suggesting that the integron linked to the antibiotic resistance wasn´t present. Using the dilution method, the most active antibiotics corresponded to the groups of cephalosporins, glycopeptides, macrolides, quinolones and tetracyclines. There was less activity of β-lactams and aminoglycosides. No variability in antibiotic profiles was recorded in isolates from different sources. According to the corrida fronts (Rf) obtained, C. pseudotuberculosis biovar ovis presented the MA types IV, V and VI. Isolates from Argentina and Brazil showed the same mycolic acid profile. CAMP and CAMP reversal tests were positive, exposing the action of exotoxin. Its apparent molecular mass was estimated at 29.55 kDa. A 100 % hemolytic activity was recorded in sheep blood, confirming the production of phospholipase D. The 3 % of the isolates did not show hemolysis on human erythrocytes and none had action on equine erythrocytes, this result could be due to the specific phospholipidic composition of the erythrocyte membrane of each mammal species. 8 The isolates showed the following enzymatic profile: 100 % showed positive production of catalase, urease, carboxyl esterase, amylase, hemolysin in ram blood and proteolytic activity. The 48 % and 97 % of the isolates showed positive the production of caseinase and hemolysin in human blood, respectively. 100 % of the isolates resulted negative in the production of lecithinase, true lipase, phenylalanine deaminase, hemolysin in horse blood and gelatinase at 4 °C and 37 °C. The results of the different trials confirmed the production of phospholipases and other enzymes and suggest the role of these virulence factors on the cytopathic effects that were observed in the cell cultures during a period of 72 h. Adherence capacity was demonstrated at 6, 24 and 72 h post-infection; and it was observed that the bacteria caused the following cytopathic effects on MDBK cells: cell lysis, cytoplasmic vacuoles, and alteration of the nucleus/cytoplasm ratio. The results of cell lysis were analyzed statistically. No significant differences were found for 6 h post-infection of the culture, while significant differences existed from 24 h onwards, with 100 % cell lysis being observed in 50 % of the isolates studied. In relation to cytoplasmic vacuolization, the statistical analysis in this case revealed significant differences from 6 hours after the beginning of the infection. The 50 % of the isolations caused the cytoplasmic vacuolization in more than 60 % of the cellular monolayer at 72 h post- infection. For the analysis of the alteration in the nucleus/cytoplasm relation, significant differences were observed among the different isolations at different times of infection. The 50 % caused the alteration in the nucleus/cytoplasm ratio in more than 40 % of the cellular monolayer at 24 h post-infection. All isolates of C. pseudotuberculosis showed the capacity to produce biofilms and the highest percentage corresponded to moderate producers (47 %). Among the isolations from Argentina, both weak (36 %) and moderate (36 %) producers predominated, while in Brazil 75 % of the isolations showed moderate biofilm production capacity and no strong producers were detected. The application of GIS allowed the results to be approached from an interdisciplinary vision. The dispersed distribution of the isolates led to their analysis considering the climate variable. This study allowed finding a relationship between the isolates coming from provinces of the Argentinean Patagonia and states of Brazil. Two well differentiated groups arose, 74 % had a direct relation with the dry climate and 26 % of the isolations were located in areas associated to the classification of rainy and temperate climate. From these results we conclude that: -There is a wide phenotypic variability among the isolations of C. pseudotuberculosis and they present differential capacities in their metabolism, antibiotic sensitivity, degrees of intensity of 9 cellular damage and of biofilm formation. Knowing these characteristics allowed establishing differential aspects and finding a climatic-seasonal link with regard to its endemic presentation in the regions of origin of the researched isolations. -C. pseudotuberculosis isolates coming from different countries correspond to the biovariety ovis. Among the Argentinian and Brazilian isolates there are biochemical variants for the production of enzymes a glucosidase and alkaline phosphatase and in the fermentation of maltose. Two biotypes are highlighted: Glu+ Gal- Malt- and Glu+ Gal- Malt+. The latter being the most frequently found at a cutaneous level and the former, in both ways: at the cutaneous level and at the visceral one. -100 % of the C. pseudotuberculosis isolates correspond to the biovariety ovis that were confirmed by using the gen sequencing that codifies the 16S rRNA sharing high probability in the level of similarity to reference strains stored in genetic banks. -A wide variety of antibiotics for public health care or veterinary usage, done by the agar diffusion method, in vitro, are quite efficient against C. pseudotuberculosis biovariety ovis. There are variable results among the isolates that develop resistance against amikacin and oxacillin. The isolates that did develop resistance against and that didn´t develop resistance against amikacin come from the Patagonia Argentinian region as well as of the Brazilian estates. -In this study, class 1 integrons were not detected in the C. pseudotuberculosis biovariety ovis isolates that developed resistance against aminoglycosides; therefore, it is possible that the resistance mechanism is given by other genetic elements. -In the in vitro dilution method (IMC), it shows a high sensitivity of C. pseudotuberculosis biovar ovis to different groups of antibiotics, but also less activity of β-lactams and aminoglycosides. -Isolates of C. pseudotuberculosis biovar ovis from different sources do not show variability in antibiotic profiles. -Despite differences in geographical location, site of lesion and animal species, C. pseudotuberculosis biovar ovis isolates from Argentina and Brazil show the same profile of mycolic acids: type IV, V and VI, corresponding to cetomycolates, epoxymicolates and dicarboxylic mycolates, respectively. -100 % of C. pseudotuberculosis biovar ovis isolates present phospholipase D, confirmed by PCR technique (pld gene) and revealed in the laboratory through CAMP and CAMP reverse biochemical tests. Both tests were key for the identification of C. pseudotuberculosis since they showed the main virulence factor of this microorganism. 10 -C. pseudotuberculosis biovariety ovis is able to adhere and invade non-phagocytic cell lines, remaining viable up to 72 h, inducing different degrees of intensity in the studied cytopathic effects. -Isolates of C. pseudotuberculosis biovar ovis, studied in this work, demonstrate virulence due to the production of some enzymes (catalase, caseinase, carboxyl esterase, amylase, urease, hemolysin), adherence capacity and internalization in non-phagocytic cell lines with production of CPE and biofilm formation. -The analysis through GIS is useful to identify the weather conditions as a variable allowing to establish a climate-seasonal link between C. pseudotuberculosis biovar ovis isolates from CL endemic areas of Argentina and Brazil.
Corynebacterium pseudotuberculosis, an etiological agent of caseous lymphadenitis (CL), is a gram-positive bacillus, pleomorphic in a coccobacillus shaped and in a palisade grouping. It is immobile, non-sporulating, non-encapsulated, facultatively anaerobic and it develops in environment enriched cultures at 37 ºC and the optimum pH at 7 to 7.2. CL is an infectious-contagious disease of chronic evolution, which mainly affects sheep, goats and other domestic animals. In horses and cows, it manifests itself as ulcerative lymphangitis and in dairy cows it can result in mastitis. CL in adult sheep can appear in cutaneous and/or visceral form. It is considered a zoonotic occupational disease worldwide. There are research studies and publications about the disease in our country as well as in Canada, Australia, France, Holland, New Zealand, United States, Brazil, Switzerland, Uruguay, Venezuela, Turkey and Cuba, among others. The objectives of this thesis were to study the morphological, physiological, metabolic and virulence factors in isolates of C. pseudotuberculosis, from various geographical areas, isolates of CL lesions in small ruminants and to determine if there is a relationship between these local isolates and other regions with high prevalence of CL. On the one hand, there were studied 35 of isolates C. pseudotuberculosis from different origins: 7 goats and 28 from sheep with different lesions and from different geographical locations. 21 separate cases from Rio Negro, Chubut, Santa Cruz and Tierra del Fuego (Argentina) and 14 isolates from the states of Sao Paulo: Aracatuba and Salvador de Bahia (Brazil). These were identified and confirmed by biochemical and molecular methods. On the other hand, the sensitivity to antibiotics was determined by agar diffusion and dilution methods. In addition, it was studied the presence of integrons containing resistance genes. Virulence factors were also studied, among them: mycolic acids, which were determined by thin-layer chromatography (TLC) of the methyl esters from mycolic acids extracted from C. pseudotuberculosis isolates. Another factor studied was exotoxin (Phospholipase D), which is the main virulence factor of this microorganism and it was determined its action through CAMP and reverse CAMP tests and through SDS-PAGE technique its apparent molecular mass was obtained. Third, the production of other extracellular enzymes and biofilm formation capacity, for which it was used a colorimetric method with violet crystal at 0.1% that allowed to classify its capacity. Through scanning electron microscopy, it was determined the interaction of a selection of C. pseudotuberculosis isolates in MDBK (Madin-Darby Bovine Kidney) cell lines and later, cytopathic effects on the cells, using Giemsa staining and optical microscopy techniques. Finally, geographic information systems (GIS) were applied as a tool for spatial analysis of C. 7 pseudotuberculosis biovari ovis isolates from different geographical sites in order to find a relationship between them. The results obtained showed that the isolates were positive for the production of catalase and urease, they also showed variable reaction for −glucosidase and alkaline phosphatase and 100 % were negative for nitrato reductase, pyrazinamidase, pyrolidonyl arylamidase, -glucuronidase, -galactosidase, N-acetyl -glucosaminidase, esculin hydrolysis and gelatin at 22 °C and 37 °C. It was determined that 100 % of the isolates from the Argentinian Patagonia and the northeast region of Brazil corresponded to the biovar ovis. There were registered biotypes in the isolates in relation to the fermentation of glucose, galactose and maltose sugars, the results determined that 33 % of the isolates corresponded to Glu+Gal-Mal- biotype and 67 % to Glu+Gal-Mal+. The 33 % of the isolates of ovine origin coming from Argentina presented the biotype Glu+Gal-Mal-. The rest of the Argentinean isolates as well as those from Brazil fermented the maltose, referring to the Glu+Gal-Mal+ biotype. This biotype was observed in 70 % of the cutaneous sample, while the biotype Glu+Gal-Mal- manifested itself in 60 % in the visceral sample. By the diffusion method, amoxicillin-clavulanic, ampicillin, cefotaxime, cefoxitin, ciprofloxacin, chloramphenicol, erythromycin, streptomycin, gentamicin, imipenem, kanamycin, norfloxacin, penicillin, rifampicin, tetracycline, trimethoprim-sulfamethoxazole and vancomycin were active in respect to 100 % of the isolates, while amikacin and oxacillin showed variable results. In the isolations that developed with amikacin and oxacillin, it was found the presence of class 1 integrons. The result was negative, suggesting that the integron linked to the antibiotic resistance wasn´t present. Using the dilution method, the most active antibiotics corresponded to the groups of cephalosporins, glycopeptides, macrolides, quinolones and tetracyclines. There was less activity of β-lactams and aminoglycosides. No variability in antibiotic profiles was recorded in isolates from different sources. According to the corrida fronts (Rf) obtained, C. pseudotuberculosis biovar ovis presented the MA types IV, V and VI. Isolates from Argentina and Brazil showed the same mycolic acid profile. CAMP and CAMP reversal tests were positive, exposing the action of exotoxin. Its apparent molecular mass was estimated at 29.55 kDa. A 100 % hemolytic activity was recorded in sheep blood, confirming the production of phospholipase D. The 3 % of the isolates did not show hemolysis on human erythrocytes and none had action on equine erythrocytes, this result could be due to the specific phospholipidic composition of the erythrocyte membrane of each mammal species. 8 The isolates showed the following enzymatic profile: 100 % showed positive production of catalase, urease, carboxyl esterase, amylase, hemolysin in ram blood and proteolytic activity. The 48 % and 97 % of the isolates showed positive the production of caseinase and hemolysin in human blood, respectively. 100 % of the isolates resulted negative in the production of lecithinase, true lipase, phenylalanine deaminase, hemolysin in horse blood and gelatinase at 4 °C and 37 °C. The results of the different trials confirmed the production of phospholipases and other enzymes and suggest the role of these virulence factors on the cytopathic effects that were observed in the cell cultures during a period of 72 h. Adherence capacity was demonstrated at 6, 24 and 72 h post-infection; and it was observed that the bacteria caused the following cytopathic effects on MDBK cells: cell lysis, cytoplasmic vacuoles, and alteration of the nucleus/cytoplasm ratio. The results of cell lysis were analyzed statistically. No significant differences were found for 6 h post-infection of the culture, while significant differences existed from 24 h onwards, with 100 % cell lysis being observed in 50 % of the isolates studied. In relation to cytoplasmic vacuolization, the statistical analysis in this case revealed significant differences from 6 hours after the beginning of the infection. The 50 % of the isolations caused the cytoplasmic vacuolization in more than 60 % of the cellular monolayer at 72 h post- infection. For the analysis of the alteration in the nucleus/cytoplasm relation, significant differences were observed among the different isolations at different times of infection. The 50 % caused the alteration in the nucleus/cytoplasm ratio in more than 40 % of the cellular monolayer at 24 h post-infection. All isolates of C. pseudotuberculosis showed the capacity to produce biofilms and the highest percentage corresponded to moderate producers (47 %). Among the isolations from Argentina, both weak (36 %) and moderate (36 %) producers predominated, while in Brazil 75 % of the isolations showed moderate biofilm production capacity and no strong producers were detected. The application of GIS allowed the results to be approached from an interdisciplinary vision. The dispersed distribution of the isolates led to their analysis considering the climate variable. This study allowed finding a relationship between the isolates coming from provinces of the Argentinean Patagonia and states of Brazil. Two well differentiated groups arose, 74 % had a direct relation with the dry climate and 26 % of the isolations were located in areas associated to the classification of rainy and temperate climate. From these results we conclude that: -There is a wide phenotypic variability among the isolations of C. pseudotuberculosis and they present differential capacities in their metabolism, antibiotic sensitivity, degrees of intensity of 9 cellular damage and of biofilm formation. Knowing these characteristics allowed establishing differential aspects and finding a climatic-seasonal link with regard to its endemic presentation in the regions of origin of the researched isolations. -C. pseudotuberculosis isolates coming from different countries correspond to the biovariety ovis. Among the Argentinian and Brazilian isolates there are biochemical variants for the production of enzymes a glucosidase and alkaline phosphatase and in the fermentation of maltose. Two biotypes are highlighted: Glu+ Gal- Malt- and Glu+ Gal- Malt+. The latter being the most frequently found at a cutaneous level and the former, in both ways: at the cutaneous level and at the visceral one. -100 % of the C. pseudotuberculosis isolates correspond to the biovariety ovis that were confirmed by using the gen sequencing that codifies the 16S rRNA sharing high probability in the level of similarity to reference strains stored in genetic banks. -A wide variety of antibiotics for public health care or veterinary usage, done by the agar diffusion method, in vitro, are quite efficient against C. pseudotuberculosis biovariety ovis. There are variable results among the isolates that develop resistance against amikacin and oxacillin. The isolates that did develop resistance against and that didn´t develop resistance against amikacin come from the Patagonia Argentinian region as well as of the Brazilian estates. -In this study, class 1 integrons were not detected in the C. pseudotuberculosis biovariety ovis isolates that developed resistance against aminoglycosides; therefore, it is possible that the resistance mechanism is given by other genetic elements. -In the in vitro dilution method (IMC), it shows a high sensitivity of C. pseudotuberculosis biovar ovis to different groups of antibiotics, but also less activity of β-lactams and aminoglycosides. -Isolates of C. pseudotuberculosis biovar ovis from different sources do not show variability in antibiotic profiles. -Despite differences in geographical location, site of lesion and animal species, C. pseudotuberculosis biovar ovis isolates from Argentina and Brazil show the same profile of mycolic acids: type IV, V and VI, corresponding to cetomycolates, epoxymicolates and dicarboxylic mycolates, respectively. -100 % of C. pseudotuberculosis biovar ovis isolates present phospholipase D, confirmed by PCR technique (pld gene) and revealed in the laboratory through CAMP and CAMP reverse biochemical tests. Both tests were key for the identification of C. pseudotuberculosis since they showed the main virulence factor of this microorganism. 10 -C. pseudotuberculosis biovariety ovis is able to adhere and invade non-phagocytic cell lines, remaining viable up to 72 h, inducing different degrees of intensity in the studied cytopathic effects. -Isolates of C. pseudotuberculosis biovar ovis, studied in this work, demonstrate virulence due to the production of some enzymes (catalase, caseinase, carboxyl esterase, amylase, urease, hemolysin), adherence capacity and internalization in non-phagocytic cell lines with production of CPE and biofilm formation. -The analysis through GIS is useful to identify the weather conditions as a variable allowing to establish a climate-seasonal link between C. pseudotuberculosis biovar ovis isolates from CL endemic areas of Argentina and Brazil.
Descripción
Palabras clave
CORYNEBACTERIUM PSEUDOTUBERCULOSIS, LINFOADENITIS CASEOSA, VIRULENCIA, BIOPELÍCULAS, ANTIBIÓTICOS
Citación
Gallardo, Adriana Alicia. (2020). Estudio de cepas de Corynebacterium pseudotuberculosis, agente etiológico de Linfoadenitis Caseosa, en pequeños rumiantes. Tesis de Doctorado. Universidad Nacional de la Patagonia San Juan Bosco.