Elina Reinoso

Virulence factors and antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis in Rio de Janeiro

Coelho S.M.O., Reinoso E., Pereira I.A., Soares L.C., Demo M., Bogni C & Souza M.M.S. 2009. Virulence factors and antimicrobial resistance in Staphylo- coccus aureus isolated from bovine mastitis in Rio de Janeiro. Pesquisa Veterinaria Brasileira 29(5):369-374. Departamento de Microbiologia e Imunologia Veterinaria, Instituto de Veterinaria, Universidade Federal Rural do Rio de Janeiro, Seropedica, RJ 23890-000, Brazil. E-mail: miliane@ufrrj.br The study was conducted to characterize pheno-genotypically the virulence factors and resistance pattern of Staphylococcus aureus isolates from milk samples of cows with subclinical mastitis. All hemolytic isolates presented beta-hemolysin, and 38% of the non- hemolytic isolates were able to express hemolysins in the presence of a beta-hemolytic strain. The amplification of the coa-gene displayed four different size polymorphisms with about 400 bp, 600 bp, 700 bp and 900 bp. The spaA gene that encodes the IgG-binding region of protein A revealed sizes of 700 bp and 900 bp. The amplification of region X from spaA yielded a single amplicon for each isolate with the prevalent amplicon size being of 180 bp. Amplification of sae gene yielded an amplicon size of 920 bp in 71% of the isolates. Antibiotic resistance pattern revealed that 42% S. aureus were susceptible to all antimicrobials tested. Seven different antibiotic patterns were observed. Our results indicated that 47% and 25% of S. aureus strains exhibited resistance to penicillin and oxacillin respectively. All oxacillin-resistant isolates were mecA-positive.

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.

Apoptosis and mutagenicity induction by a characterized aqueous extract of Baccharis articulata (Lam.) Pers. (Asteraceae) on normal cells

In a previous study we have demonstrated that cold aqueous extract of Baccharis articulata (Ba-CAE) induced the death of human peripheral blood mononuclear cells (PBMCs) and exerted low mutagenic effects on mice at 6h after administration. The aim of this work was to investigate whether the PBMCs death induced by Ba-CAE is due to apoptosis, and whether this extract exerts mutagenic effects on mice at 24 and 48h after administration. In addition, Ba-CAE was chemically characterized. PBMCs from healthy volunteers were exposed to extract (10, 20, 40, 80, 160, 320, 640 and 1280μg/mL) for 18-24h. Cell viability was determined by staining of trypan blue dye exclusion method. Apoptosis was determined by Hoechst 33258 staining, TUNEL, and DNA fragmentation analysis by agarose gel electrophoresis. BALB/c mice were injected with extract (1800, 900 and 450mg/kg) and sacrificed at 24 and 48h postinjection. Bone marrow samples were used to assess chromosome mutations by the micronucleus test. The extract induced PBMCs death by apoptosis and increased the frequency of micronuclei in bone marrow. The phytochemical study of Ba-CAE showed the presence of flavones as luteolin and acacetin, caffeoylquinic acids as chlorogenic acid, and tannins.

Evaluation of the cytotoxicity, genotoxicity and apoptotic induction of an aqueous extract of Achyrocline satureioides (Lam.) DC

Achyrocline satureioides is widely consumed as infusion or aperitif and shows important therapeutic properties. Previously, we reported absence of genotoxicity of cold aqueous extract (CAE) of A. satureioides by Allium test. However, one test cannot predict the genotoxic effects of a substance. Thus, the aim of this work was to investigate cytotoxicity, genotoxicity and apoptotic ability of CAE of A. satureioides. In addition, CAE was chemically characterized. The cytotoxicity was evaluated by Trypan blue and MTT assays. The apoptotic capacity was evaluated by Hoechst staining and DNA fragmentation-analysis. The genotoxicity was studied by comet assay (CA) and micronucleus test. The identification and quantification of flavonoids were performed by HPLC-ESI-MS/MS. The cytotoxicity studies indicated low toxicity of CAE. In addition, CAE did not induce apoptotic effects on human PBMCs. CAE did not show genotoxicity in vitro against Vero cells, at 10-50 μg/mL. CAE did not induce in vivo genotoxic effects, but it showed at high concentrations cytotoxicity by micronucleus assay. CAE presented flavonoids such as quercetin, 3-O-methylquercetin and luteolin. In conclusion, A. satureioides at popularly concentrations used, in aperitif or infusion, can be consumed safely because did not show any cytotoxic or genotoxic effects.

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.

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.

Phenotyping and genotyping of streptococci in bovine milk in Argentinean dairy herds.

Most veterinary and milk hygiene laboratories identify streptococci and enterococci based on serological and biochemical tests. The analysis of 16S rDNA was suggested to be used for more exact identification; however, its use has not been considered so far in monitoring studies. The objective of the present study was to compare a conventional phenotypic method with restriction fragment length polymorphism analysis of 16S rDNA (16S rDNA RFLP) for identification of streptococci isolated from composite milk samples collected in connection with intramammary infection (IMI) in six Argentinean dairy farms. Composite milk samples (n = 1223) from cows belonging to six herds were collected for bacteriological analysis. Twelve reference strains and fifty streptococci or streptococcuslike isolates were identified to species level by the API 20 Strep system, conventional biochemical tests and 16S rDNA RFLP in a blind assay. The remaining streptococci or streptococcus-like isolates (n = 40) were identified to the species level both by 16S rDNA RFLP and conventional biochemical tests. As indicated by Kappa values, agreement between the 16S rDNA RFLP and the conventional scheme for identification of Streptococcus agalactiae, S. dysgalactiae, S. uberis, S. equinus and Enterococcus faecalis was 0.91, 0.73, 0.92, 0.81 and 0.85, respectively. Together with the less frequently isolated streptococcal species, the conventional scheme correctly identified 77 out of 90 isolates (85.5%). Thus, the use of 16S rDNA RFLP is considered valuable for monitoring studies due to its affordable cost for standard laboratories.

Lack of cytotoxic and genotoxic effects of Minthostachys verticillata essential oil: Studies in vitro and in vivo

Minthostachys verticillata (peperina) is an aromatic and medicinal plant with several uses and ethnobotanical properties. Numerous studies have demonstrated that its essential oil (Mv-EO) presents antimicrobial capacity and shows immunomodulating and anti-allergic properties in human cell lines. Thus, the goal of this study was to investigate the main chemical composition, analyzed by GC-FID, and the cyto-genotoxic effects of Mv-EO, using Vero cells, human PBMCs and mice bone marrow cells. The Mv-EO was rich in pulegone 60.5% and menthone 18.2%. Our results clearly show that Mv-EO is not cyto-genotoxic in vitro nor in vivo. It not induced cytotoxic effects, as indicated by trypan blue dye exclusion and NRU assays both in Vero cells and human PBMCs. In addition, Mv-EO (100-1000 μg/mL) not induced apoptotic effects on human PBMCs, as indicated by Hoechst staining and DNA fragmentation analysis by agarose gel electrophoresis. The in vivo assay showed that Mv-EO (25-500 mg/kg) not increased the frequency of micronucleus in bone marrow cells of mice. Further, the ratio of polychromatic/normochromatic erythrocytes was not modified. These findings suggest that Mv-EO appears to be safe as a therapeutic agent.

In Vitro and In Vivo Cytogenotoxic Effects of Hot Aqueous Extract of Achyrocline satureioides (Lam.) DC.

In this work we extend the toxicological studies of hot aqueous extract of A. satureioides (As-HAE) evaluating cytotoxic and apoptotic effects on human peripheral blood mononuclear cells (PBMCs). We also determine genotoxic action of this extract in vivo. In addition, the extract was chemically characterized. Finally, we established a comparison with previous data of cold aqueous extract. The As-HAE induced cytotoxicity on PBMCs determined by trypan blue dye exclusion (CC50 = 653 μg/mL) and MTT (CC50 = 588 μg/mL) assays being more toxic than cold extract. However, As-HAE as well as cold extract did not induce apoptosis measured by Hoechst 33258 staining, TUNEL assay, and DNA fragmentation analysis. The in vivo micronucleus test showed that As-HAE exerted cytogenotoxic effects on bone marrow of mice, contrary to what was observed with cold extract. The chemical study of As-HAE allowed identifying the flavonoids found in cold extract: luteolin, quercetin, and 3-O-methylquercetin, but at higher concentrations. We suggest that toxic effects induced by As-HAE could be due to high concentrations of these flavonoids. Given that As-HAE is the most used in folkloric medicine, its administration should be controlled in order to prevent potential cell damage.