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)

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.

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.