Short communication: Extended-spectrum AmpC-producing Escherichia coli from milk and feces in dairy farms in Brazil.

The AmpC enzyme is normally expressed constitutively in Escherichia coli, and its overproduction confers resistance to cefoxitin. A newly reported AmpC, the extended-spectrum AmpC (ESAC), is related to resistance to cefepime, a fourth-generation cephalosporin. This enzyme presents more flexibility in the active site due to insertions, replacements, and deletions on AA sequences. Many isolates producing ESAC were reported in human clinical isolates, but E. coli ESAC producers were reported in animals only in France. The animal E. coli strains can produce this enzyme and possibly disseminate it to human and production environments. In our study, 3 strains of E. coli from milk and feces bovine samples, collected in Rio de Janeiro, Brazil, were suspected to produce ESAC. After excluding other mechanisms of resistance, the gene was sequenced to verify ESAC characteristics. These strains presented replacement of AA in omega and R2 loops, suggesting ESAC production. This is the first report to study ESAC E. coli in dairy farms in Brazil.

Characterization of Coagulase-Negative Staphylococci and pheno-genotypic beta lactam resistance evaluation in samples from bovine Intramammary infection / Caracterização de Staphylococcus coagulase negativa e avaliação fenogenotípica da resistência aos beta-lactâmicos em amostras oriundas de infecção intra-mamária em bovinos

This study aimed to identify Coagulase-Negative Staphylococci (CoNS) species isolated from bovine mastitis, through phenotypic and PCR-RFLP (Restriction Fragment Length Polymorphism-Polimerase Chain Reaction) methods and to compare both techniques to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique. Among them, the PCR-RFLP method, using a partially conserved sequence of the groEL gene, is a promising alternative, because of its reproducibility and reliability. On the other hand, the proteomic technique MALDI-TOF MS provides an accurate and much faster diagnosis and has been increasingly employed in microbiological identification. The pheno-genotypic profiles of beta-lactam resistance were also investigated, this characterization is important, considering that the use of antimicrobials is a key element for mastitis control in dairy farms. The concordance of the phenotypic, PCR-RFLP and MALDI-TOF MS assays to identify CoNS species was 77,5% (31/40). S. chromogenes was the species most frequently isolated. Antibiotic resistance rate was relatively low, registering values of 25.5% to penicillin, 9.6% to oxacillin and 6.2% to cefoxitin. Resistance to imipenem, cephalotin and amoxicillin+clavulanate was not observed. The mecA gene and its variant were detected in 7.6% and 4,1% of the isolates respectively. The blaZ gene was found in 43.2% of the strains resistant to penicillin.(AU)

Este estudo teve como objetivo identificar isolados de Staphylococcus coagulase-negativa (SCN) isolados de mastite bovina, por meio de métodos fenotípicos e PCR-RFLP (reação em cadeia de polimerase - polimorfismo nos fragmentos de restrição), e compará-los com a técnica de tempo de voo de ionização/desorção por laser assistida por matriz de espectrofotometria de massa (MALDI-TOF MS). O método de PCR-RFLP, que utiliza uma parte conservada da sequência do gene groEL, é uma alternativa promissora, por ser reprodutível e confiável. Por outro lado, a técnica proteômica MALDI-TOF MS permite uma acurácia e um diagnóstico muito mais rápidos e tem sido cada vez mais empregada na identificação microbiológica. Os perfis fenogenotípicos de resistência aos beta-lactâmicos também foram investigados. Essa caracterização é importante, considerando-se que os antimicrobianos são os elementos-chave para o controle da mastite na produção leiteira. A concordância entre os testes fenotípicos, PCR-RFLP E MALDI-TOF MS na identificação foi de 77,5% (31/40). S. chromogenes foi a espécie mais frequentemente isolada. A resistência antimicrobiana foi relativamente baixa, apresentando valores de 25,5% para penicilina, 9,6% para oxacilina e 6,2% para cefoxitina. Resistência ao imipenem, à cefalotina e à amoxacilina + ácido clavulâncico não foi observada. O gene mecA e sua variante foram detectados em 7,6% e 4,1% dos isolados, respectivamente. O gene blaZ foi encontrado em 43,2% dos isolados resistentes à penicilina.(AU)

Characterization of virulence and antibiotic profile and agr typing of Staphylococcus aureus from milk of subclinical mastitis bovine in State of Rio de Janeiro / Caracterização do perfil de virulência e resistência antimicrobiana e tipagem agr de Staphylococcus aureus oriundos do leite bovino de mastite subclinica no estado do Rio de Janeiro

This study aims to detect the main virulence and antimicrobial resistance genes in Stapylococcus aureus from bovine mastitic milk as well as classifying them according to agr typing. A total of 55 strains from six dairy unities in the state of Rio de Janeiro were selected, of these 27.3% presented fbnA and 78,2% for fbnB genes, respectively. None of the strains tested were positive for cap5 gene, 3.6% were positive for cap8 gene. Additionally, 94.5% of strains had hlA gene and 89.1% had hlB gene while 67.3% of the strains had icaA gene and 87.3% had icaD gene. From these results it was possible to establish 12 different virulence profiles. Prevalence of agrII type was detected in 81.8% of the isolates. Concerning antimicrobial resistance evaluation, the studied strains were susceptible to all antibiotics tested except penicillin, 83.6% being resistant strains. None of the strains had mecA gene, however, 40% of the strains had blaZ gene. Associating virulence and resistance data made it possible to obtain 23 different profiles. This great diversity of strains shows wide array of bacterial strategies and the challenge of mastitis prevention in cattle. Despite antimicrobial susceptibility, these strains presented certain genes that allow its persistence in the herd.(AU)

O presente estudo teve como objetivo detectar os principais genes de virulência e resistência antimicrobiana em Staphylococcus aureus oriundos de leite bovino mastítico e classificá-los de acordo com a tipagem do gene agr. Foram selecionados 55 isolados de seis unidades produtores no estado do Rio de Janeiro. Destas, o gene fbnA foi encontrado em 27,3% das cepas e 78,2% possuíam o gene fbnB. Em nenhuma cepa foi encontrado o gene cap5 e 3,6% possuíam o gene cap8. O gene hlA foi encontrado em 94,5% das cepas e 89,1% possuíam o gene hlB. O gene icaA foi encontrado em 67,3% das cepas e 87,3% possuíam o gene icaD. Com base nesses resultados, foi possível estabelecer 12 diferentes perfis de virulência. Prevalência do agr tipo II foi detectada em 81,8% dos isolados. Considerando-se a avaliação da resistência antimicrobiana, as cepas estudadas foram suscetíveis a todos os antibióticos exceto penicilina, sendo detectado um percentual de 83,6% de cepas resistentes. Nenhuma das cepas apresentou o gene mecA, contudo 40% das cepas apresentaram o gene blaZ. Vinte e três perfis diferentes foram estabelecidos por associação de dados de virulência e resistência. Essa grande diversidade de cepas mostra a ampla gama de estratégias bacterianas e o desafio da prevenção à mastite no gado bovino, considerando-se que, a despeito da suscetibilidade antimicrobiana, essas cepas apresentam genes que permitem sua persistência no rebanho.(AU)

Perfil clínico e microbiológico de cães com e sem otoacaríase / Clinical and microbiological profile of dogs with and without otoacariasis

Os objetivos do estudo foram identificar a presença de microrganismos nos condutos auditivos dos cães através dos exames citológico e microbiológico, assim como avaliar a associação destes à otoacaríase e seus sinais clínicos. O diagnóstico da infestação por Otodectes cynotis foi realizado através de otoscopia bilateral e pela coleta de material e visualização do parasito sob microscópio esterioscópico, constituindo o exame parasitológico. Outras coletas de secreção otológica de cada orelha foram realizadas para confecção de lâminas para a citologia e para o isolamento microbiológico, sendo este último material coletado através de "swab" estéril. Dos 250 animais examinados, em 15 (6%) foi identificado o ácaro O. cynotis.Observou-se associação entre a presença do parasito e a ocorrência de otite clínica externa caracterizada especialmente por eritema auricular (P<0,001) e a produção excessiva de secreção otológica (P=0,0016), assim como a observação, pelos proprietários, de prurido ótico nos cães (P<0,001). Sugere-se a possibilidade de essas associações serem efeito da infestação. Houve também a associação (P<0,01) entre a otoacaríase e a ocorrência de microrganismos nos dois condutos auditivos de cada animal, detectados através da citologia. No entanto, não houve associação (P=0,04 e p=0,07) entre a presença do ácaro O. cynotis e a ocorrência de microrganismos no exame microbiológico das orelhas direita e esquerda, respectivamente.

The goals of this study were to identify microorganisms in dogs' ear canals through cytological and microbiological examination, and evaluate their association with otoacariasis and its clinical signs. Otodectes cynotis infestation diagnosis was achieved by bilateral otoscopy, and by parasite viewing on cerumem under stereoscopic microscope, representing the parasitological exam. Ear discharge was also collected from each canal to fix cytology slides and for microbiological isolation, but this last sample was collected with sterile swab. Among the 250 studied animals, 15 (6%) had ear mites. There was an association between the parasite and external clinical otitis specially as established by auricular erythema (P<0,001) and excessive production of ear discharge (P=0,0016), as well as ear pruritus observed by the owners (P<0,001). It has been suggested that these associations can be an effect of the infestations. There was also an association (P<0,01) between otoacariasis and microorganisms detected by cytological exam in both ear canals of each dog. However, there was no association (P=0,04 e P=0,07) between the presence of ear mite and microorganisms detected by microbiological exam of right and left ears.

Adição da parede celular de levedura (Saccharomyces cerevisiae) na dieta para frangos de corte / Addition of the yeast cell wall (Saccharomyces cerevisiae) in the diet for broilers chickens

Os efeitos da parede celular de Saccharomyces cerevisiae (PCSc) na dieta para frangos foram avaliados com base nos tratamentos: dieta referência com avilamicina (AV); dieta referência (DR); DR com 0,1% de PCSc (PCSc0,1); DR com 0,2% de PCSc (PCSc0,2); DR com 0,3% de PCSc (PCSc0,3). O delineamento foi o de blocos ao acaso, com cinco tratamentos e seis repetições de 10 aves, totalizando 300 frangos, machos. No período de nove a 21 dias de idade, o tratamento PCSc0,3 influenciou negativamente no ganho de peso (631g), e no período de 34 a 39 dias, o tratamento PCSc0,1 proporcionou melhor ganho de peso (461g) em relação ao tratamento com o antimicrobiano. O maior peso vivo ao abate, 2571g, foi obtido no tratamento PCSc0,1. Os pesos absolutos de sobrecoxa e coxa+sobrecoxa foram maiores nos tratamentos PCSc0,1 (290g e 560g) e PCSc0,2 (292g e 561g) e menores no tratamento PCSc0,3 (263g e 515g). Não houve influência na metabolizabilidade da ração e na contagem total de coliformes totais no íleo. Dietas com inclusão de até 0,2% de PCSc resultaram em índices produtivos adequados, podendo ser utilizadas como aditivo em dietas livres de antimicrobiano melhorador de desempenho sem o comprometimento do desempenho, características de carcaça, metabolizabilidade da ração e da contagem total de coliformes totais do íleo.

The effects of the yeast cell wall (Saccharomyces cerevisiae) (CWSc) in the diet of broiler chickens were evaluated through the following treatments: reference diet with avilamicin (AV); reference diet (RD); RD with 0.1% CWSc (CWSc0,1); RD with 0.2% CWSc (CWSc0,2); RD with 0.3% CWSc (CWSc0,3). The study design was randomized blocks, with five treatments and six replicates of 10 birds, totaling 300 male broilers. In the period from 9 to 21 days treatment CWSc0.3 had a negative influence on weight gain (631g) and from 34 to 39 days CWSc0.1 provided better weight gain (461g) compared to the treatment with the antimicrobial. The highest live weight value at slaughter, 2,571 grams, was obtained in treatment CWSc0.1. The absolute weights of drumstick and thigh+drumstick were higher in treatments CWSc0.1 (290g and 560g) and CWSc0.2 (292g and 561g) and were lower for CWSc0.3 (263g and 515g). There was no influence of the treatments on both the metabolizability and the total count of coliforms in the ileum. Diets until 0.2% of PCSc inclusion resulted in correct productive rates and they can be used as a ration growth promoter additive in antimicrobial free diets without damage on performance, carcass characteristics, organ weight, metabolizability of the diets and total coliform count in the ileum.

Selection of lactic acid bacteria to promote an efficient silage fermentation capable of inhibiting the activity of Aspergillus parasiticus and Fusarium gramineraum and mycotoxin production.

AIMS: To select lactic acid bacteria with potential silage inoculant properties. The bio-control activity against mycotoxicogenic fungi and the presence of antibiotics resistance gene were also evaluated. METHODS AND RESULTS: Lactobacillus rhamnosus RC007 and Lactobacillus plantarum RC009 were selected on the basis of growth rate and efficacy in reducing the pH of maize extract medium; therefore, they were evaluated for their bio-control ability against Fusarium graminearum and Aspergillus parasiticus. Studies on lag phase, growth rate and aflatoxin B1 (AFB1) and zearalenone (ZEA) production were carried out in vitro under different regimes of aw (0·95 and 0·99); pH (4 and 6); temperature (25 and 37°C); and oxygen availability (normal and reduced). Lactobacillus rhamnosus RC007 was able to completely inhibit the F. graminearum growth at all assayed conditions, while Lact. plantarum RC009 only did it at pH 4. Both Lactobacillus strains were able to significantly reduce the A. parasiticus growth rate mainly at 0·99 aw . A decrease in ZEA production was observed as result of Lactobacillus strains -F. graminearum interaction; however, the A. parasiticus- Lact. plantarum interaction resulted in an increased AFB1 production. Lactobacillus rhamnosus RC007 proved to have no genes for resistance to the tested antibiotics. CONCLUSIONS: The ability of Lact. rhamnosus RC007 to rapidly drop the pH and to inhibit fungal growth and mycotoxin production and the absence of antibiotic resistance genes shows the potential of its application as inoculant and bio-control agent in animal feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of selecting bacteria for silage inoculants not only for the improvement of silage fermentation but also for their effects on mycotoxicogenic fungi and the resulting mycotoxin production due to the risk that they may involve.

Short communication: profile of virulence factors of Staphylococcus aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil.

Staphylococcus aureus produces exoproteins that contribute to its ability to colonize the mammary gland such as hemolysins, coagulase, slime, and protein A. This study characterized phenotypically and genotypically these virulence factors in 50 Staph. aureus isolates. These isolates were obtained from milk samples from subclinical mastitis cases identified in 15 dairy cattle farms located in the state of Rio de Janeiro, Brazil. All of the confirmed Staph. aureus samples were PCR positive for the coa gene, which displayed 3 different size polymorphisms. The amplification of the spaA X region yielded a single amplicon for each isolate with the prevalent amplicon sized 315 bp. The Staph. aureus isolates were 24 and 16% positive for the hla and hlb genes, respectively, and 22 and 20% positive for the icaA and icaD genes, respectively. Amplification of the agr gene RNAIII was positive in 74% of the strains. Twenty-seven different profiles were identified among the samples, indicating a great diversity of Staph. aureus involved in the etiology of mastitis cases in the analyzed region. These findings are valuable to the comprehension of the distribution of the profiles of Staph. aureus strains isolated from subclinical mastitis cases in the state of Rio de Janeiro.

Resistência bacteriana e ação das bacteriocinas de Lactobacillus spp em Staphylococcus aureus isolados de mastite bovina / Bacterial resistance and bacteriocins action in Staphylococcus aureus isolated from bovine mastitis

Staphylococcus aureus is the major pathogen causing intramammary infections in dairy cattle worldwide. Among the factors that contribute to its spread and infectious potential is the ability to overcome the mechanisms of antimicrobials activity. The present work investigated the antimicrobial resistance pattern and sensibility to bacteriocins produced by strains of Lactobacillus spp of 30 isolates of S. aureus from mastitis. From this, 29 are beta-lactamase producers. Eight isolates (26.6 percent) showed resistance to at least four antibiotics being considered multiresistent. All of them were mecA-positive. Otherwise, all isolates tested showed sensibility to at least one of the four bacteriocin producer strains. Due to the significant depletion of the efficacy of antimicrobials, pathogen growth inhibition by bacteriocins seems an alternative of biological control in infectious processes.

Caracterização genotípica de amostras Escherichia coli isoladas de mastite bovina / Genotypic characterization of Escherichia coli isolated from cows with mastitis

Escherichia coli samples isolated from cases of dairy mastitis in municipalities of Rio de Janeiro State, Brazil, were genotypically compared and Shiga-toxin genes were detected and their prevalence evaluated. Genetically related samples were verified and the referred genes were detected in 14.28% of the cases.

Enterobacterial microbiota on Stomoxys calcitrans external surface.

The current study evaluated the prevalence of enterobacterial agents on the external surface of the stable fly, Stomoxys calcitrans. In addition, this study investigated the presence of virulence genes of enterobacteria. Twenty different species were isolated and identified, Escherichia coli was the most frequent species isolated. The genes stx1, stx2 and/or eae for production of Shiga toxin were present in 13.04% of the E. coli samples.

Suscetibilidade à azitromicina de agentes bacterianos isolados de processos infecciosos em diferentes sítios de animais de companhia / Azithromycin susceptibility pattern of bacterial isolated from different sites of infections in pet animals

Avaliou-se o perfil de suscetibilidade bacteriana de diferentes sítios infecciosos frente aos antimicrobianos de eleição e determinaram-se o perfil de atividade in vitro e a concentração inibitória mínima (CIM) da azitromicina. Diferentes testes fenotípicos detectaram resistência à azitromicina em 45 por cento de Staphylococcus spp. e 65 por cento dos bastonetes Gram-negativo. A CIM50 para S. aureus foi 4,0μg/mL para S. intermedius 1,0μg/mL, Staphylococcus spp. coagulase-negativo >512μg/mL e bastonetes Gram-negativo 256μg/mL. Investigou-se, também, uma possível resistência cruzada entre oxacilina e azitromicina por meio da detecção do gene mecA em Staphylococcus spp. Foi possível detectar resistência à azitromicina em nove (15 por cento) isolados de Staphylococcus spp. mecA positivo.

Antimicrobials susceptibility pattern of bacterial isolated from different sites of infection, in vitro azithromycin activity pattern, and its minimum inhibitory concentration (MIC) values were evaluated. Different phenotypic tests detected azithromycin resistance in 45 percent of Staphylococcus spp. and 65 percent of resistant Gram-negative rods. MIC50 was 4.0μg/mL for Staphylococcus aureus, 1.0μg/mL for S. intermedius, >512.0μg/mL for coagulase negative Staphylococcus, and 256.0μg/mL for Gram-negative rods. In addition, it was investigated the possible cross-resistance between oxacillin and azithromycin, by detection of mecA gen in Staphylococcus spp. Nine (15 percent) mecA-positive Staphylococcus spp. were also phenotypically resistant to azithromycin.

Microbiota bacteriana em segmentos de mosca do estábulo Stomoxys calcitrans no Brasil: primeiro relato de espécies / Microbiota in Stomoxys calcitrans segments in Brazil: first description of species

This study report the first description of bacterial species recovered from body segments of Stomoxys calcitrans collected in dairy farms from Rio de Janeiro State, Brazil. Eighteen out of 33 isolated and identified species were unknown in stable flies by the worldwide literature. Some of these species have the potential to develop diseases in man and other animals and some others have not been described yet as pathogenic agents.