Different distribution of antimicrobial resistance genes and virulence profiles of Staphylococcus aureus strains isolated from clinical mastitis in six countries.

Staphylococcus aureus is recognized worldwide as one of the main contagious mastitis agents in cattle and can express a set of antimicrobial resistance genes and virulence-associated genes that explain the wide range of outcomes of intramammary infections. Staphylococcus aureus strains are heterogeneous: their different resistance and virulence patterns, associated with host-level factors and treatment factors, are related to the severity of infection. The aim of this study was to determine phenotypic antibiotic susceptibility, occurrence of selected antimicrobial resistance genes and other virulence genes in 93 S. aureus strains isolated from clinical mastitis in 6 countries: Argentina, Brazil, Germany, Italy, the United States (New York State), and South Africa. These isolates were tested against a total of 16 drugs (amoxicillin-clavulanate, ampicillin, cefazolin, cefoperazone, cefquinome, enrofloxacin, erythromycin, gentamicin, kanamycin, lincomycin, oxacillin, penicillin, rifampin, spiramycin, sulfamethoxazole/trimethoprim, tylosin) by minimum inhibitory concentration (MIC) assay, and examined for the presence of 6 antibiotic-resistance genes (blaZ, mecA, mecC, ermA, ermB, ermC) and 6 virulence-associated genes (scn, chp, sak, hla, hlb, sea) via PCR analysis. The phenotypic results of this study revealed the presence of 19.4% penicillin-resistant strains, whereas 22.6% of the strains were classified as having resistance (5.4%) or intermediate resistance (17.2%) to erythromycin. Most (96.8%) of the isolates were inhibited by cephalosporins, and all were susceptible to amoxicillin-clavulanate. Two strains (1 from Germany, 1 from Italy) were resistant to oxacillin and were positive for mecA. Among the other antimicrobial resistance genes, the most frequently detected was blaZ (46.2%), and 32.3% of the isolates were positive for erm genes: ermC (21.5%) and ermB (10.8%). The most prevalent virulence gene was hla (100%), followed by hlb (84.9%) and sea (65.6%). These results show a low prevalence of antibiotic multidrug resistance in S. aureus isolates, even if the detection of selected antimicrobial resistance genes did not always correspond with the occurrence of phenotypic antibiotic resistance; the immune evasion cluster gene prevalence was quite low in the samples analyzed.

Standardization of bulb and root sample sizes for the Allium cepa test.

Although the Allium cepa test has been widely used to identify potentially cytotoxic and genotoxic pollutants in aquatic environments, variable non-standardized choices have been made regarding the number of plant bulbs and roots analyzed. We propose numbers for bulbs and roots per bulb when comparing the frequencies of micronuclei, mitotic anomalies and mitotic index with this test. Roots that had been treated with aqueous solutions, such as water samples collected in August 2007 from the Paraíba do Sul River at the Brazilian cities of Tremembé and Aparecida; negative and positive controls were used for bioassays. The presence of pollutants in the river water had been presumed based on our previous cytological data and an official report by the São Paulo State Environmental Agency (Brazil) on presence of fecal contaminants (Tremembé and Aparecida) and elevated dissolved aluminium (Aparecida) in the water under study. The sampling of ten bulbs and five roots per bulb was found adequate for comparative studies to evaluate with the A. cepa test the potential damage inflicted by pollutants in aquatic environments. Furthermore, even one bulb and one root per bulb was sufficient in discerning this damage, thereby shortening the time required to attain a statistically confident comparative evaluation. However, to allow for the use of statistical programs based on the evaluation of average values, and to avoid criticism based on genetic variability, we propose that three bulbs and three roots per bulb be considered as standard sample sizes for the A. cepa test.

Evaluation of the cytotoxic and genotoxic potential of water from the River Paraíba do Sul, in Brazil, with the Allium cepa L. test.

This work investigated the cytotoxic and genotoxic potential of water from the River Paraíba do Sul (Brazil) using Allium cepa roots. An anatomo-morphological parameter (root length), mitotic indices, and frequency of micronuclei were analysed. Eight bulbs were chosen at random for treatment for 24 to 120 hours with the River water collected in the years of 2005 and 2006 from sites in the cities of Tremembé and Aparecida (São Paulo state, Brazil). Daily measurements of the length of the roots grown from each bulb were carried out throughout the experiment. Mitotic index (MI) and frequency of micronuclei (MN) were determined for 2000 cells per root, using 3-5 root tips from other bulbs (7-10). Only in the roots treated with samples of the River water collected in 2005 in Tremembé city was there a decrease in the root length growth compared to the respective control. However, a reduction in MI values was verified for both sites analysed for that year. Considering the data involving root length growth and especially MI values, a cytotoxic potential is suggested for the water of the River Paraíba do Sul at Tremembé and Aparecida, in the year of 2005. On the other hand, since in this year the MN frequency was not affected with the river water treatments, genotoxicity is not assumed for the river water sampled at the aforementioned places.

Evaluation of the cytotoxic and genotoxic potential of water from the River Paraíba do Sul, in Brazil, with the Allium cepa L. test / Avaliação do potencial citotóxico e genotóxico da água do Rio Paraíba do Sul através do teste Allium cepa

This work investigated the cytotoxic and genotoxic potential of water from the River Paraíba do Sul (Brazil) using Allium cepa roots. An anatomo-morphological parameter (root length), mitotic indices, and frequency of micronuclei were analysed. Eight bulbs were chosen at random for treatment for 24 to 120 hours with the River water collected in the years of 2005 and 2006 from sites in the cities of Tremembé and Aparecida (São Paulo state, Brazil). Daily measurements of the length of the roots grown from each bulb were carried out throughout the experiment. Mitotic index (MI) and frequency of micronuclei (MN) were determined for 2000 cells per root, using 3-5 root tips from other bulbs (7-10). Only in the roots treated with samples of the River water collected in 2005 in Tremembé city was there a decrease in the root length growth compared to the respective control. However, a reduction in MI values was verified for both sites analysed for that year. Considering the data involving root length growth and especially MI values, a cytotoxic potential is suggested for the water of the River Paraíba do Sul at Tremembé and Aparecida, in the year of 2005. On the other hand, since in this year the MN frequency was not affected with the river water treatments, genotoxicity is not assumed for the river water sampled at the aforementioned places.

Este trabalho é parte de uma investigação sobre o potencial citotóxico e genotóxico da água do Rio Paraíba do Sul (Brasil) utilizando raízes de Allium cepa. Foi analisado um parâmetro anátomo-morfológico (crescimento das raízes), bem como o índice mitótico e a frequência de micronúcleos. Aleatoriamente oito bulbos foram submetidos aos tratamentos de 24 a 120 horas com água do Rio proveniente dos pontos de coleta das cidades de Tremembé e Aparecida nos anos de 2005 e 2006. O comprimento das raízes de cada bulbo foi acompanhado diariamente ao longo do experimento. O índice mitótico (IM) e a frequência de micronúcleos (MN) foram determinados pela análise de 2.000 células por raiz, sendo utilizadas 3-5 raízes de outros bulbos (7-10). Somente as raízes tratadas com amostras de água coletadas em 2005 na cidade de Tremembé, apresentaram decréscimo no comprimento das raízes quando comparadas com o controle. Entretanto, foi observada redução do IM nas raízes tratadas com água de ambos os pontos de coleta no mesmo ano. Considerando os dados de crescimento de raiz e especialmente IM, um potencial citotóxico é sugerido para a água do Rio Paraíba do Sul em Tremembé e Aparecida, no ano de 2005. Por outro lado, para este mesmo ano, a frequência de micronúcleos não foi alterada; assim, a genotoxicidade não foi assumida para a água do Rio nos pontos mencionados.

Evaluation of the cytotoxic and genotoxic potential of water from the River Paraíba do Sul, in Brazil, with the Allium cepa L. test

This work investigated the cytotoxic and genotoxic potential of water from the River Paraíba do Sul (Brazil) using Allium cepa roots. An anatomo-morphological parameter (root length), mitotic indices, and frequency of micronuclei were analysed. Eight bulbs were chosen at random for treatment for 24 to 120 hours with the River water collected in the years of 2005 and 2006 from sites in the cities of Tremembé and Aparecida (São Paulo state, Brazil). Daily measurements of the length of the roots grown from each bulb were carried out throughout the experiment. Mitotic index (MI) and frequency of micronuclei (MN) were determined for 2000 cells per root, using 3-5 root tips from other bulbs (7-10). Only in the roots treated with samples of the River water collected in 2005 in Tremembé city was there a decrease in the root length growth compared to the respective control. However, a reduction in MI values was verified for both sites analysed for that year. Considering the data involving root length growth and especially MI values, a cytotoxic potential is suggested for the water of the River Paraíba do Sul at Tremembé and Aparecida, in the year of 2005. On the other hand, since in this year the MN frequency was not affected with the river water treatments, genotoxicity is not assumed for the river water sampled at the aforementioned places.

Este trabalho é parte de uma investigação sobre o potencial citotóxico e genotóxico da água do Rio Paraíba do Sul (Brasil) utilizando raízes de Allium cepa. Foi analisado um parâmetro anátomo-morfológico (crescimento das raízes), bem como o índice mitótico e a frequência de micronúcleos. Aleatoriamente oito bulbos foram submetidos aos tratamentos de 24 a 120 horas com água do Rio proveniente dos pontos de coleta das cidades de Tremembé e Aparecida nos anos de 2005 e 2006. O comprimento das raízes de cada bulbo foi acompanhado diariamente ao longo do experimento. O índice mitótico (IM) e a frequência de micronúcleos (MN) foram determinados pela análise de 2.000 células por raiz, sendo utilizadas 3-5 raízes de outros bulbos (7-10). Somente as raízes tratadas com amostras de água coletadas em 2005 na cidade de Tremembé, apresentaram decréscimo no comprimento das raízes quando comparadas com o controle. Entretanto, foi observada redução do IM nas raízes tratadas com água de ambos os pontos de coleta no mesmo ano. Considerando os dados de crescimento de raiz e especialmente IM, um potencial citotóxico é sugerido para a água do Rio Paraíba do Sul em Tremembé e Aparecida, no ano de 2005. Por outro lado, para este mesmo ano, a frequência de micronúcleos não foi alterada; assim, a genotoxicidade não foi assumida para a água do Rio nos pontos mencionados.