Relative expression of genes associated with adhesion to bovine mammary epithelial cells by Streptococcus uberis.

Streptococcus uberis is one of the most prevalent environmental pathogens of bovine mastitis. Biofilm growth ability by S. uberis looks to depend first upon the adherence of cells to a surface. The S. uberis ability to adhere to mammary gland epithelia might provide an advantage to colonize the lactating mammary gland. The objectives of this study were (a) to select S.uberis strains according to their ability to form biofilm, (b) to determine adherence to and internalization into MAC-T cells and (c) to investigate the expression profile adherence genes in these S. uberis strains. For the assays, the MAC-T bovine mammary epithelial cell line was used. Relative expression of genes acdA, lmb, scpA, sua, fbp and lbp was quantified by RT-qPCR. We observed that the RC38 strain from clinical bovine mastitis showed in the six genes higher values than control in both conditions. While the strain with greater ability to adhere, from clinical mastitis and biofilm producer (RC29) evidenced higher values in group 1 (G1) (bacteria after the initial contact with MAC-T cells) and decrease in group 2 (G2) (both adhered and internalized bacteria) than control. Strains with a moderate or strong capacity for biofilm production showed significantly lower relative expression values in the G2. In all adherence associated genes, strain RC19 showed relative expression values incremented in G1, while in G2 decreased expression. In conclusion, we did not find a single profile of relative expression because the relative expression levels of each gene differed depending on the strain and the co-culture stage of S. uberis cells from which RNA was obtained.

Fibronectin and laminin induce biofilm formation by Streptococcus uberis and decrease its penicillin susceptibility.

The aim of this study was to determine the effect of fibronectin and laminin on the in vitro biofilm formation by Streptococcus uberis and the susceptibility to penicillin under planktonic and biofilm growth conditions. We observed that a high percentage (76.5%) of the S. uberis isolates was weak biofilm producers in Todd Hewitt Broth (THB). A high percentage of moderate (38.2%) or strong (53%) biofilm producers was observed in THB supplemented with laminin or fibronectin, respectively. All S. uberis isolates growing as planktonic cells were sensitive to penicillin. Minimum biofilm inhibitory concentrations (MBICs) were ranging between 0.25 and 2 µg/ml, whereas minimum biofilm eradication concentrations (MBECs) ranging from 8 to 256 µg/ml. These results show that biofilm-growing S. uberis cells required higher concentrations of the antibiotic than those needed to inhibit planktonic cells. Similar MBICs of penicillin were obtained when S. uberis cells growing in THB supplemented or not with laminin or fibronectin, whereas the MBECs markedly increased when one of two proteins were added to culture medium compared with the medium without proteins. To the best of our knowledge, this is the first report of decreased susceptibility to penicillin likely related to a higher production of biofilms stimulated by laminin or fibronectin. Therapeutic failures of penicillin to treat S. uberis infections may be due to biofilm formation.

Genotyping and study of adherence-related genes of Streptococcus uberis isolates from bovine mastitis.

The aim of this study was to determine the presence, conservation, and distribution of 6 potential adherence genes and their relationship with diverse molecular types in 34 S. uberis isolated from bovine mastitis in Argentina. Pulsed-field gel electrophoresis (PFGE) typing with SmaI was performed. The PCR for the detection of each gene, scpA, acdA, fbp, lbp, lmb, and sua was standardized. Samples of the amplification products were purified and sequenced. The PFGE patterns revealed the high level of heterogeneity of S. uberis, with 26 types of PFGE patterns. A high prevalence of scpA, fbp, lbp, lmb and acdA genes (100%-97%) was detected, whereas 79.41% of S. uberis harbored the sua gene. A high degree of similarity in the nucleotide and amino acid sequences of the 6 genes was observed. Our results showed that all genes are conserved and are present in most S. uberis isolates despite the wide clonal heterogeneity detected. This is the first study reporting an analysis of prevalence, and nucleotides and amino acids sequences of the potential adherence genes scpA, acdA, fbp, lbp, and lmb from S. uberis strains versus reported GenBank sequences, S. uberis 0140J and S. uberis NZ01.

Streptococcus uberis: In vitro biofilm production in response to carbohydrates and skim milk / Streptococcus uberis: producción in vitro de biofilm en respuesta a carbohidratos y leche descremada

Streptococcus uberis has become one of the most important environmental pathogens associated with clinical and subclinical bovine mastitis. Biofilm confers to bacteria more resistance to physical and chemical agents as well as to different mechanisms of the innate immune system. The aim of this work was to evaluate the ability of in vitro biofilm production in 32 S. uberis isolates from bovine mastitis and identified by biochemical tests and milk subsequently confirmed by the amplification of the pauA gene. The isolates were cultivated in TMP broth and TMP broth with the addition of 0.5% glucose, 1% sucrose, 1% lactose or 0.5% skim milk in microtiter plates stained with crystal violet. We demonstrated that S. uberis isolated from bovine mastitis are able to produce biofilms in TMP broth and, also that biofilm formation by S. uberis can be significantly enhanced by the addition of 0.5% glucose or 1% sucrose to TMP broth. This may suggest that the carbohydrates in milk or within the ruminant gut might affect the growth mode of S. uberis. In addition, our results showed that in vitro biofilm production under different conditions of supplementation displays variation among the isolates and that each isolate shows a particular profile of biofilm production. This phenotypic heterogeneity in biofilm production exhibited by S. uberis could at least partly explain why this bacterium has the ability to adapt to different niches facilitating survival to diverse and stressful conditions.

Streptococcus uberis es uno de los más importantes patógenos medioambientales asociados a la mastitis bovina clínica y subclínica. El biofilm confiere a las bacterias resistencia a agentes físicos y químicos, como así también a diferentes mecanismos del sistema inmune innato. El objetivo del presente estudio fue evaluar la habilidad de producción de biofilm in vitro de 32 aislamientos de S. uberis recuperados de mastitis bovina, previamente identificados por pruebas bioquímicas y confirmados por la amplificación del gen pauA. Los aislamientos fueron cultivados en caldo TMP sin carbohidratos, y además en caldo TMP con la adición de 0,5% de glucosa, 1% de sacarosa, 1% de lactosa o 0,5% de leche descremada, en placas de microtitulación tenidas con cristal violeta. Se demostró que dichos aislamientos son capaces de producir biofilm en caldo TMP, y además se observó un incremento significativo en la producción de biofilm en caldo TMP suplementado con 0,5% de glucosa o con 1% de sacarosa. Así, los carbohidratos de la leche o los presentes dentro del intestino de los rumiantes podrían afectar el modo de crecimiento de S. uberis. Además, nuestros hallazgos mostraron que la producción de biofilm in vitro en diferentes condiciones de suplementación presenta variabilidad entre los aislamientos de S. uberis y que cada aislamiento muestra un perfil particular de producción de biofilm. Esta heterogeneidad fenotípica en la producción de biofilm de S. uberis podría explicar, al menos en parte, por qué esta bacteria tiene la habilidad de adaptarse a diferentes nichos, lo que le facilita la supervivencia frente a condiciones diversas y estresantes.

Streptococcus uberis: In vitro biofilm production in response to carbohydrates and skim milk.

Streptococcus uberis has become one of the most important environmental pathogens associated with clinical and subclinical bovine mastitis. Biofilm confers to bacteria more resistance to physical and chemical agents as well as to different mechanisms of the innate immune system. The aim of this work was to evaluate the ability of in vitro biofilm production in 32 S. uberis isolates from bovine mastitis and identified by biochemical tests and subsequently confirmed by the amplification of the pauA gene. The isolates were cultivated in TMP broth and TMP broth with the addition of 0.5% glucose, 1% sucrose, 1% lactose or 0.5% skim milk in microtiter plates stained with crystal violet. We demonstrated that S. uberis isolated from bovine mastitis are able to produce biofilms in TMP broth and, also that biofilm formation by S. uberis can be significantly enhanced by the addition of 0.5% glucose or 1% sucrose to TMP broth. This may suggest that the carbohydrates in milk or within the ruminant gut might affect the growth mode of S. uberis. In addition, our results showed that in vitro biofilm production under different conditions of supplementation displays variation among the isolates and that each isolate shows a particular profile of biofilm production. This phenotypic heterogeneity in biofilm production exhibited by S. uberis could at least partly explain why this bacterium has the ability to adapt to different niches facilitating survival to diverse and stressful conditions.

Prevalence and antibiotic susceptibility of coagulase-negative Staphylococcus species from bovine subclinical mastitis in dairy herds in the central region of Argentina.

Coagulase-negative staphylococci (CNS) are a common cause of bovine subclinical mastitis (SCM). The prevalence of CNS species causing SCM identified by genotyping varies among countries. Overall, the antimicrobial resistance in this group of organisms is increasing worldwide; however, little information exists about a CNS species resistant to antibiotics. The aim of the present study was to genotypically characterize CNS at species level and to determine the prevalence and antibiotic resistance profiles of CNS species isolated from bovine SCM in 51 dairy herds located in the central region of the province of Cordoba, Argentina. In this study, we identified 219 CNS isolates at species level by PCR-restriction fragment length polymorphism of the groEL gene. Staphylococcus chromogenes (46.6%) and Staphylococcus haemolyticus (32%) were the most prevalent species. A minimum of three different CNS species were present in 41.2% of the herds. S. chromogenes was isolated from most of the herds (86.3%), whereas S. haemolyticus was isolated from 66.7% of them. The broth microdilution method was used to test in vitro antimicrobial susceptibility. Resistance to a single compound or two related compounds was expressed in 43.8% of the isolates. S. chromogenes and S. haemolyticus showed a very high proportion of isolates resistant to penicillin. Resistance to two or more non-related antimicrobials was found in 30.6% of all CNS. S. haemolyticus exhibited a higher frequency of resistance to two or more non-related antimicrobials than S. chromogenes.

Prevalencia y sensibilidad a antibióticos de especies de estafilococos coagulasa negativos provenientes de mastitis subclínica en bovinos de tambos de la región central de Argentina / Prevalence and antibiotic susceptibility of coagulase-negative Staphylococcus species from bovine subclinical mastitis in dairy herds in the central region of Argentina

Coagulase-negative staphylococci (CNS) are a common cause of bovine subclinical mastitis (SCM). The prevalence of CNS species causing SCM identified by genotyping varies among countries. Overall, the antimicrobial resistance in this group of organisms is increasing worldwide; however, little information exists about a CNS species resistant to antibiotics. The aim of the present study was to genotypically characterize CNS at species level and to determine the prevalence and antibiotic resistance profiles of CNS species isolated from bovine SCM in 51 dairy herds located in the central region of the province of Cordoba, Argentina. In this study, we identified 219 CNS isolates at species level by PCR-restriction fragment length polymorphism of the groEL gene. Staphylococcus chromogenes (46.6%) and Staphylococcus haemolyticus (32%) were the most prevalent species. A minimum of three different CNS species were present in 41.2% of the herds. S. chromogenes was isolated from most of the herds (86.3%), whereas S. haemolyticus was isolated from 66.7% of them. The broth microdilution method was used to test in vitro antimicrobial susceptibility. Resistance to a single compound or two related compounds was expressed in 43.8% of the isolates. S. chromogenes and S. haemolyticus showed a very high proportion of isolates resistant to penicillin. Resistance to two or more non-related antimicrobials was found in 30.6% of all CNS. S. haemolyticus exhibited a higher frequency of resistance to two or more non-related antimicrobials than S. chromogenes.

Los estafilococos coagulasa negativos (ECN) son una causa frecuente de mastitis subclínica (MSC) en bovinos. La prevalencia de especies de ECN causantes de MSC identificadas por métodos genotípicos varía entre países. La resistencia antimicrobiana en este grupo de organismos se está incrementando en el mundo; sin embargo, existe poca información acerca de las especies de ECN resistentes a antibióticos. Los objetivos del presente estudio fueron caracterizar genotípicamente los ECN a nivel de especie y determinar la prevalencia y los perfiles de resistencia a antibióticos de las especies de ECN aisladas de MSC en bovinos de 51 rodeos situados en la provincia de Córdoba, Argentina. Mediante polimorfismos de los fragmentos de restricción del gen groEL identificamos 219 aislamientos de ECN a nivel de especie. Staphylococcus chromogenes (46,6%) y Staphylococcus haemolyticus (32%) fueron las especies más prevalentes. Un mínimo de 3 especies diferentes de ECN estuvieron presentes en el 41,2% de los tambos. S. chromogenes fue aislado en la mayoría de los tambos (86,3%), mientras que S. haemolyticus fue aislado en el 66,7% de aquellos. Para el análisis de sensibilidad a los antimicrobianos in vitro se usó el método de microdilución en caldo. La resistencia a un único compuesto o a 2 compuestos relacionados fue expresada en el 43,8% de los aislamientos. S. chromogenes y S. haemolyticus mostraron una muy elevada proporción de aislamientos resistentes a penicilina. La resistencia a 2 o más antimicrobianos no relacionados fue hallada en el 30,6% de los ECN. S. haemolyticus exhibió una frecuencia de resistencia a 2 o más antimicrobianos no relacionados más elevada que S. chromogenes.