Differentiation of non-aureus staphylococci species isolated from bovine mastitis by PCR-RFLP of groEL and gap genes in comparison to MALDI-TOF mass spectrometry.

Intramammary infections (IMI) cause serious economic losses for farmers and the dairy industry. Cases of subclinical mastitis are commonly the result of infection by minor pathogens such as non-aureus staphylococci (NAS), so their correct identification is important for appropriate therapeutic intervention and management. The aim of this study was to assess the reliability of PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) of the groEL and gap genes to discriminate between bovine-associated NAS species, using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as the reference method. MALDI-TOF MS was able to correctly identify 112 NAS isolates from bovine IMI at species level out of a total of 115 (97.4%). These results were considered definitive and thus compared with those from the PCR-RFLP analyses. Only 50% (56/112) of the samples classified through groEL PCR-RFLP matched the molecular identity determined by MALDI-TOF MS, whereas coincidence rose to 96.4% (108/112) when comparing results from gap PCR-RFLP and the spectral analysis. This study demonstrates that gap PCR-RFLP is a useful and reliable tool for the identification of NAS species isolated from bovine mastitis.

Screening of bacteriocin associated genes of Streptococcus uberis strains.

A wide variety of contagious and environmental bacteria can cause bovine mastitis worldwide. Antibiotic therapy is currently used for the treatment of the disease, although its intensive use leads to the emergence of resistant strains. Bacteriocins arise as potential antibacterial option for mastitis treatment. The aim of this work was to analyze bacteriocin associated genes as Streptococcus uberis ubericin A (ubaA), ubericin A immunity protein (ubaI), uberolysin A (ublA), Lantibiotic nisin-U (nsuA and nsuB) in 68 S uberis strains. Furthermore, the ability of the strains to inhibit important mastitis pathogens was assayed. Results showed that genes were present in combination and all the strains carried at least one gene. Seven bacteriocion associated gene patterns were identified. S. uberis strains were able to inhibit different mastitis pathogens and the greatest inhibition was observed in CNS strains. The results obtained provide new insights on antibacterial activity produced by S. uberis strains against different mastitis pathogens and could contribute to the development of strategies to treat intramammary infections.

Perfiles genéticos de Streptococcus uberis aislados de mastitis bovina / Genetic patterns of Streptococcus uberis isolated from bovine mastitis

El objetivo del presente estudio fue evaluar las relaciones genotípicas entre 40 Streptococcus uberis aislados de mastitis bovina mediante la técnica de electroforesis de campos pulsantes (pulsed-field gel electrophoresis [PFGE]). Además, se investigó la asociación entre los patrones de PFGE y los perfiles de virulencia. Los aislamientos mostraron 17 patrones de PFGE. Se encontraron diferentes cepas dentro de los tambos y en los distintos tambos, y un bajo número de aislamientos dentro del mismo tambo compartieron un perfil idéntico de PFGE. No se encontró ninguna asociación entre los patrones de PFGE y los perfiles de virulencia. Sin embargo, la detección de cepas particulares en algunos tambos podría indicar que algunas de ellas son más virulentas que otras. Sería importante avanzar en las investigaciones para identificar nuevos genes relacionados con la virulencia que podrían contribuir a la capacidad infecciosa de estas cepas

The aim of this study was to evaluate the genotypic relationships among 40 Streptococcus uberis isolated from bovine mastitis by using pulsed-field gel electrophoresis (PFGE). Additionally, the association between PFGE patterns and virulence profiles was investigated. The isolates exhibited 17 PFGE patterns. Different strains were found within and among herds; however, a low number of isolates within the same herd shared an identical PFGE type. No association between PFGE patterns and virulence profiles was found. However, the detection of specific strains in some herds could indicate that some strains are more virulent than others. Further research needs to be undertaken to elucidate new virulence-associated genes that might contribute to the capability of these strains to produce infection

Genetic patterns of Streptococcus uberis isolated from bovine mastitis.

The aim of this study was to evaluate the genotypic relationships among 40 Streptococcus uberis isolated from bovine mastitis by using pulsed-field gel electrophoresis (PFGE). Additionally, the association between PFGE patterns and virulence profiles was investigated. The isolates exhibited 17 PFGE patterns. Different strains were found within and among herds; however, a low number of isolates within the same herd shared an identical PFGE type. No association between PFGE patterns and virulence profiles was found. However, the detection of specific strains in some herds could indicate that some strains are more virulent than others. Further research needs to be undertaken to elucidate new virulence-associated genes that might contribute to the capability of these strains to produce infection.

Análise fenotípica e genotípica da virulência de Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina / Phenotypic and genotypic analysis of virulence in Staphylococcus spp. and its clonal dispersion as a contribution to the study of bovine mastitis

A mastite é uma inflamação da glândula mamária causada principalmente por bactérias, dentre as quais o gênero Staphylococcus ocupa um papel importante. Bactérias pertencentes a este gênero são caracterizadas por expressar fatores de virulência que permitem sua persistência e disseminação no hospedeiro. O presente trabalho teve por objetivo avaliar fenogenotipicamente os fatores de virulência de isolados de Staphylococcus spp. a partir de casos de mastite bovina. Foram analisadas 272 amostras de leite provenientes de oito propriedades da região Sul-Fluminense do Estado do Rio de Janeiro. Após identificação, obteve-se um total de 250 isolados de Staphylococcus spp. Estes foram submetidos às provas fenotípicas de detecção da produção de "slime" em microplaca e em ágar vermelho congo; produção de hemolisinas e sinergismo hemolítico; produção de caseinase e DNase. Posteriormente foram submetidos à técnica de PCR para detecção dos genes de produção de cápsula (cap5 e cap8), fibronectina (fnbA,e fnbB), "slime" (icaA e icaD) e hemolisinas (hla e hlb). Do total avaliado, 58% (145/250) foi identificado como Staphylococcus spp. coagulase-negativos e 42% (105/250) como Staphylococcus spp. coagulase-positivos, destes 36,2% (38/105) foram identificados como S. aureus, 11,4% (12/105) como S. intermedius e 3,8% (4/105) como pertencentes ao grupo SIG. Apenas 6,4% (16/250) dos isolados foram produtores de α-hemólise, 4,8% (12/250) de β-hemólise e, 1,6% (4/250) de α e β-hemólise. A produção de caseinase foi observada em 66,4% (166/250), e a produção de "slime" avaliada pela técnica da microplaca em 76,8% (192/250) dos isolados, respectivamente. A DNase foi detectada em ECNs (38/145) e S. aureus (14/38). Os marcadores genéticos avaliados para a produção de slime, icaA e icaD apresentaram nenhuma ou leve concordância com a produção fenotípica, respectivamente, utilizando o coeficiente Kappa. Tal dado parece indicar que outros marcadores genéticos podem estar envolvidos com a expressão desta característica. Os demais genes detectados com frequência de 4% (10/250) para cap5 e para cap8, 32,8% (82/250) para fnbA, 4,4% (11/250) para fnbB, 19,2% (48/250) para hla e 18% (45/250) para hlb. O perfil circulante nas propriedades foi o 1: isolado produtor de "slime" e caseinase. O gene spaA foi positivo em todos os S. aureus, apresentando amplicons de tamanhos variados, sendo o tamanho prevalente o de 300pb. A amplificação do gene coa apresentou nove tipos polimórficos distintos, sendo prevalente o amplicon de 600pb. O gene agr foi detectado em todos os S. aureus, com amplicon de 200pb. Foi observado que os genes de virulência estudados estavam distribuídos de modo aleatório entreos 6 distintos perfis eletroforéticos obtidos através da Eletroforese em Gel de Campo Pulsado (PFGE).

Mastitis is an inflammation of one or more mammary glands caused mainly by bacteria, among which the genus Staphylococcus plays an important role. Bacteria belonging to this genus are known to express virulence factors which allow their persistence and spread in the host. This study aimed to evaluate the phenotypic and genotypic aspects of virulence factors in Staphylococci spp. isolates from bovine mastitis clinical cases. A total of 272 milk samples from 8 farms in the South-Fluminense region of Rio de Janeiro were analyzed. The samples underwent conventional bacterial identification, yielding 250 Staphylococci spp. isolates. These were tested for the phenotypic detection of slime production by the microplate and Congo Red Agar methods. The hemolysins production, hemolytic synergism, caseinase and DNase production were also evaluated. The isolates were then assayed through the Polymerase Chain Reaction method to detect genes associated with virulence factors such as: capsule (cap5, cap8), fibronectin (fnbA, fnbB), slime (icaA, icaD) and hemolysins (hla e hlb). Regarding the number of isolates assessed, 58% (145/250) were identified as coagulase-negative Staphylococcus spp. and 42% (105/250) as coagulase-positive Staphylococcus spp. The latter comprised 36.2% (38/105) of isolates identified as S. aureus, 11.4% (12/105) as S. intermedius and 3.8% (4/105) belonging to the SIG group. The hemolisin production was not significant, whereas only 6,4% (16/250) produced alfa hemolysis, 4,8% (12/250) produced beta hemolysis and 1,6% (4/250) was able to produce both. Caseinase production was observed in 66.4% (166/250) and slime production assayed through the microplate method was positive in 76,8% (192/250). DNAse was detected in coagulase-negative Staphylococcus spp. (38/145) and in S. aureus (14/38). Low association between genetic detection of icaA (38/250) and icaD (54/250) and slime phenotypic expression (192/250) suggest that others genetic markers can be involved in this expression. Regarding gene amplification, the isolates did not show significant correlation between the genetic detection of icaA (38/250) and icaD (54/250) and slime production (192/250), indicating that other genetic markers may be involved in this trait expression. The frequency of the occurrence of the others studied genes was of 4% (10/250) for cap5 and cap8, 32,8% (82/250) for fnbA, 4,4% (11/250) for fnbB, 19,2% (48/250) for hla and 18% (45/250) for hlb. The major circulating strain profile on the farms encompassed slime and caseinase producer strains. The spaA gene was found in all of the S. aureus isolates, presenting varying amplicons sizes, with 300bp being the prevalent size. The amplification of the coa gene showed nine polymorphic variants, with 600bp being the prevalent amplicon. The agr gene was also detected in every S. aureus isolate, with an amplicon of 200bp. It was noticed that the presence or absence of the virulence genes assayed in this study were not correlated with the 6 distinct electrophoretic profiles obtained by PFGE.

Development of an experimentally induced Streptococcus uberis subclinical mastitis in goats.

Streptococcus uberis is a major environmental mastitis-causing pathogen. The infections are predominantly subclinical and are frequently undetected and untreated for extended periods of time. More information about the pathogenesis of S. uberis mastitis would be useful. To our knowledge, no experimental studies into the mastitis pathogenesis caused by S. uberis have been described in lactating goats. The aim of this study was to reproduce an experimentally induced S. uberis subclinical mastitis in lactating goats aimed to evaluate the inflammatory response, dynamics of infection and the pathological findings within the first hours of intramammary inoculation with S. uberis. Six Saanen goats in mid-lactation were inoculated with 1.7 × 10(8)cfu of S. uberis. Bacterial growth peaked in milk from challenged right mammary halves (RMH) at 4h PI. Shedding of viable bacteria showed a marked decrease at 20 h PI. Mean somatic cell counts in milk from the RMH peaked at 20 h PI. Inoculation with S. uberis was followed by a decrease in the mean total number of leukocytes. Signs and systemic symptoms were not evoked by intramammary inoculation. S. uberis could be isolated in tissue from all RMH. Histological examination of specimens of the RMH and lymph nodes of the goats showed an increased inflammatory response throughout the experiment. The histological findings correlated with the immunohistochemical detection of S. uberis in RMH. In conclusion, the experimental inoculation of S. uberis in lactating goats is capable of eliciting an inflammatory response and causing pathological changes, resulting in a subclinical mastitis. This investigation shows that goat might to represent a valuable model for the study of the mastitis pathogenesis caused by S. uberis.

Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine subclinical mastitis in Argentinean dairy farms

The aim of this study was to investigate the phenotypic and genotypic characteristics of Streptococcus uberis isolated from subclinical mastitis (SCM) cases, and to examine the possible association between both characteristics. A total of 32 S. uberis were isolated from 772 quarter milk samples (SCM > 250,000 cells/ml) collected from 195 cows selected randomly from 18 dairy farms located in Argentina. The S. uberis strains were characterized phenotypically by the presence of virulence factors as plasminogen activator factor (PAF), hyaluronidase (HYA), capsule (CAP) and CAMP factor, and were further characterized genotypically by pulsed-field gel electrophoresis (PFGE). S. uberis strains expressed plasminogen activator factor, hyaluronidase or capsule (65.5 %, 56.3 %, 59.4 %, respectively), but only 25 % of isolates were CAMP factor positive. Thirteen different virulence profiles were identified on the basis of the combination of virulence factors. Eighteen PFGE patterns with 90% of similarity were identified among 32 S. uberis. A great diversity of virulence profiles and PFGE patterns were present among dairy farms. S. uberis strains with the same PFGE pattern showed different virulence profiles. Bovine S. uberis strains causing SCM included in the present study showed heterogeneity in regard to their phenotypic and genotypic characteristics, and the PFGE patterns are not associated with the virulence profiles.

Caracterización fenotípica y genotípica de Streptococcus uberis aislados de mastitis bovina subclínica en tambos de Argentina. El objetivo de este estudio fue investigar las características fenotípicas y genotípicas de Streptococcus uberis aislados de casos de mastitis subclínica (MSC) y examinar la posible asociación entre ambas características. Un total de 32 cepas de S. uberis fueron aisladas de 772 muestras de leche de cuartos mamarios (MSC > 25 0000 células/ml) colectadas de 195 vacas seleccionadas al azar pertenecientes a 18 tambos lecheros localizados en Argentina. Las cepas de S. uberis fueron caracterizadas fenotípicamente sobre la base de la presencia de factores de virulencia tales como el factor activador del plasminógeno (FAP), la hialuronidasa (HIA), la cápsula (CAP) y el factor CAMP. Además, fueron caracterizadas genotípicamente por electroforesis de campos pulsados (PFGE). Las cepas de S. uberis expresaron el factor activador del plasminógeno, la hialuronidasa o la cápsula (65,5 %, 56,3 % y 59,4 %, respectivamente), pero solo el 25 % fueron CAMP positivas. Sobre la base de la combinación de los factores de virulencia se identificaron 13 perfiles de virulencia diferentes. Asimismo, se identificaron 18 patrones de PFGE con un 90 % de similitud entre las 32 cepas de S. uberis. Se presentó una gran diversidad de perfiles de virulencia y patrones de PFGE entre los tambos. Cepas con el mismo patrón de PFGE presentaron perfiles de virulencia diferentes. Las cepas de S. uberis causantes de MSC en bovinos incluidas en el presente estudio mostraron heterogeneidad con respecto a sus características fenotípicas y genotípicas, y los patrones de PFGE no estuvieron asociados con los perfiles de virulencia.

Distribution of virulence-associated genes in Streptococcus uberis isolated from bovine mastitis.

Streptococcus uberis is an important pathogen that has been implicated in bovine mastitis but the virulence factors associated with pathogenesis are not well understood. The aim of this work was to examine 11 putative and known virulence-associated genes by PCR in 78 S. uberis strains isolated from infected animals in Argentina. Additionally, the distribution of virulence patterns over various herds was determined. Not all genes were present in the strains but all of the detected virulence-associated genes were present in combination. Forty-seven (60.3%) isolates carried seven to 10 virulence-associated genes. Further analysis revealed 58 virulence patterns. Different patterns were found within the same herd and among herds, demonstrating that strains with different virulence patterns were able to cause mastitis. Despite the large number of strains with different virulence patterns, strains with identical patterns was found. Detection of virulence-associated genes in individual S. uberis strains isolated from infected animals revealed one to 10 virulence genes. This may indicate that other virulence factors could be involved. The present study reveals the occurrence and distribution of 11 virulence-associated genes among S. uberis isolates from bovine mastitis in various herds and contributes to a better understanding of the pathogenicity of this bacterium.

Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine subclinical mastitis in Argentinean dairy farms.

The aim of this study was to investigate the phenotypic and genotypic characteristics of Streptococcus uberis isolated from subclinical mastitis (SCM) cases, and to examine the possible association between both characteristics. A total of 32 S. uberis were isolated from 772 quarter milk samples (SCM > 250,000 cells/ml) collected from 195 cows selected randomly from 18 dairy farms located in Argentina. The S. uberis strains were characterized phenotypically by the presence of virulence factors as plasminogen activator factor (PAF), hyaluronidase (HYA), capsule (CAP) and CAMP factor, and were further characterized genotypically by pulsed-field gel electrophoresis (PFGE). S. uberis strains expressed plasminogen activator factor, hyaluronidase or capsule (65.5 %, 56.3 %, 59.4 %, respectively), but only 25 % of isolates were CAMP factor positive. Thirteen different virulence profiles were identified on the basis of the combination of virulence factors. Eighteen PFGE patterns with 90% of similarity were identified among 32 S. uberis. A great diversity of virulence profiles and PFGE patterns were present among dairy farms. S. uberis strains with the same PFGE pattern showed different virulence profiles. Bovine S. uberis strains causing SCM included in the present study showed heterogeneity in regard to their phenotypic and genotypic characteristics, and the PFGE patterns are not associated with the virulence profiles.

Método para cuantificar actividad proteolítica sobre sustratos insolubles coloreados / Method for quantitative proteolytic activity about stained substrates insoluble

Se cuantificó la actividad proteolítica de Staphylococcus aureus aislados de infecciones del hombre, aplicando una modificación de la técnica efectuada por otros autores con Pseudomonas aeruginosa. Además del polvo de piel coloreado con Azul Brillante de Remazol utilizado con esta bacteria, se prepararon otros sustratos insolubles, tales como elastina-Rojo Congo, colágeno-Rojo Congo y polvo de ubre-Rojo Congo. Fue posible comprobar mayor actividad proteolítica sobre colágeno que sobre elastina, mientras que con los extractos o polvos tisulares se evidenció más proteólisis con polvo de ubre de vaca (PUH) que con polvo de piel (PPA). Cabe destacar que las cepas procedían de infecciones humanas, incluyendo afecciones no epidérmicas y una de colección ATCC. El método desarrollado constituyó una ventaja con respecto a las técnicas clásicas de bacteriología que detectan cualitativamente la actividad proteolítica

Método para cuantificar actividad proteolítica sobre sustratos insolubles coloreados / Method for quantitative proteolytic activity about stained substrates insoluble

Se cuantificó la actividad proteolítica de Staphylococcus aureus aislados de infecciones del hombre, aplicando una modificación de la técnica efectuada por otros autores con Pseudomonas aeruginosa. Además del polvo de piel coloreado con Azul Brillante de Remazol utilizado con esta bacteria, se prepararon otros sustratos insolubles, tales como elastina-Rojo Congo, colágeno-Rojo Congo y polvo de ubre-Rojo Congo. Fue posible comprobar mayor actividad proteolítica sobre colágeno que sobre elastina, mientras que con los extractos o polvos tisulares se evidenció más proteólisis con polvo de ubre de vaca (PUH) que con polvo de piel (PPA). Cabe destacar que las cepas procedían de infecciones humanas, incluyendo afecciones no epidérmicas y una de colección ATCC. El método desarrollado constituyó una ventaja con respecto a las técnicas clásicas de bacteriología que detectan cualitativamente la actividad proteolítica