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Staphylococcus aureus is an important pathogen involved in subclinical bovine mastitis, causing high economic losses for the dairy industry. The successful persistence of this pathogen in the host occurs due to a series of factors associated with colonization ability and the acquisition of virulence factors. This bacterial species carries genetic heterogeneity, and genetically diverse strains characterize the population. Analysis of the genetic variation is an important tool for epidemiological studies. For this study, S. aureus strains were randomly selected by molecular profiling. All strains were originated from the milk of cows of subclinical mastitis on farms in the State of Rio de Janeiro. Strains of S. aureus were profiled using virulence gene analysis profiles, agr and spa typing, Pulsed Field Gel Electrophoresis (PFGE), and Multilocus Sequence Typing (MLST). 47% (8/17) of the tested strains were positive strains for icaA gene; 82.3% (14/17) for icaD gene; 41% (7/17) for fbnA gene; 47% (8/17) positive for fbnB gene; 94% (16/17) for the hlA gene and 70.5% (12/17) for hlB gene. These virulence results generated 11 different profiles. Most strains (58.8% - 10/17) were classified as type-II by agr system. spa typing identified seven different spa types. PFGE analysis found extensive genetic heterogeneity and no clones were observed. MLST analysis generated five different types of ST/CC. Considering the results observed at the present study, a high genetic variety of S. aureus strains associated of the presence of different virulence factors justified the absence of clonal strains at the properties evaluated. Besides, that, the definition of S. aureus clonal strain as well dynamic population in not fully understood since there are a limited number of studies in S. aureus associated with bovine mastitis., Staphylococcus aureus é um importante patógeno envolvido na mastite subclínica bovina, causando altas perdas econômicas para a indústria de laticínios. A persistência bem-sucedida desse patógeno no hospedeiro ocorre devido a uma série de fatores associados à capacidade de colonização e aquisição de fatores de virulência. Esta espécie bacteriana possui heterogeneidade genética e a população é caracterizada por cepas geneticamente diversas. A análise da variação genética é uma ferramenta importante para estudos epidemiológicos. Para este estudo, cepas de S. aureus foram selecionadas aleatoriamente por perfil molecular. Todas as cepas foram originadas do leite de vacas de mastite subclínica em fazendas do Estado do Rio de Janeiro. As cepas de S. aureus foram caracterizadas usando perfis de análise de genes de virulência, tipagem agr e spa, eletroforese em gel de campo pulsado (PFGE) e tipagem de sequência de foco múltiplo (MLST). 47% (8/17) das cepas testadas foram positivas para o gene icaA; 82.3% (14/17) para o gene icaD; 41% (7/17) para o gene fbnA; 47% (8/17) positivo para o gene fbnB; 94% (16/17) para o gene hlA e 70.5% (12/17) para o gene hlB. Esses resultados de virulência geraram 11 perfis diferentes. A maioria das cepas (58.8% - 10/17) foi classificada como tipo II pelo sistema agr. A tipificação por spa identificou sete tipos diferentes. A análise por PFGE encontrou extensa heterogeneidade genética e nenhum clone foi observado. A análise do MLST gerou cinco tipos diferentes de ST / CC. Considerando os resultados observados no presente estudo, uma elevada variedade genética de cepas de S. aureus associada à presença de diferentes fatores de virulência justificou a ausência de cepas clonais nas propriedades avaliadas. Além disso, a definição de cepas clonais assim como a dinâmica populacional em S. aureus não é totalmente compreendida, pois há um número limitado de estudos em S. aureus associados à mastite bovina.

The contribution of the animal environments to the worsening of the global antimicrobial resistance framework is related to the use of antimicrobials in subtherapeutic doses and, for long periods, establishing ideal conditions for the circulation of resistance genes, which can be transmitted to pathogens adapted to the human microbiota. The study of the animal environment as conducive to the acceleration of resistance evolution is an emerging and critical area for understanding the development and dissemination of resistance genes among the circulating bacteria. The connection between people, animals, and the environment allows us to consider antimicrobial resistance in an approach within the “One Health” concept, which provides a global strategy for expanding collaboration and interdisciplinary communication. This chapter will highlight the emergence of colistin resistance, a great challenge in antimicrobial resistance field. Also, it will focus on some agents included in the priority list of superbugs of the World Health Organization (WHO) or correlated species already identified in veterinary medicine, such as the critical superbugs; priority level 1, Carbapenem-resistant Acinetobacter baumannii, Carbapenem-resistant Pseudomonas aeruginosa, and ESBL-producing Carbapenemic-resistant Enterobacteriaceae; and the high-priority, level 2, methicillin-resistant Staphylococcus aureus (MRSA).

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. RESUMO 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.

The prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) as a cause of infectious disease in companion animals remains unknown. The emergence of MRSP is a challenge in veterinary medicine as multidrug-resistant strains began to emerge, resulting in treatment failures. This study provides an overview of the characterization of S. pseudintermedius strains from clinical pet samples and the prevalence of MRSP strains. A total of 123 S. pseudintermedius strains were characterized by phenotypic testing and the MALDI-TOF technique and evaluated for susceptibility to methicillin and the presence of the mecA gene. Of these, 49 (39.8%) were identified as MRSP. The results confirm the importance of monitoring resistant pathogens and the need for further studies to determine the prevalence of MRSP in companion animals. The prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) as a cause of infectious disease in companion animals remains unknown. The emergence of MRSP is a challenge in veterinary medicine as multidrug-resistant strains began to emerge, resulting in treatment failures. This study provides an overview of the characterization of S. pseudintermedius strains from clinical pet samples and the prevalence of MRSP strains. A total of 123 S. pseudintermedius strains were characterized by phenotypic testing and the MALDI-TOF technique and evaluated for susceptibility to methicillin and the presence of the mecA gene. Of these, 49 (39.8%) were identified as MRSP. The results confirm the importance of monitoring resistant pathogens and the need for further studies to determine the prevalence of MRSP in companion animals., A prevalência de Staphylococcus pseudintermedius (MRSP) resistente à meticilina como causadores de doenças infecciosas em animais de companhia permanece desconhecida. O surgimento de MRSP é um desafio na medicina veterinária, já que cepas resistentes a múltiplas drogas começaram a surgir, resultando em falhas no tratamento. Este estudo fornece uma visão geral sobre a caracterização de cepas de S. pseudintermedius oriundas de amostras clínicas de animais de companhia e a prevalência de cepas de MRSP. Um total de 123 cepas de S. pseudintermedius foram caracterizados através de provas fenotípicas e pela técnica de MALDI-TOF e avaliadas quanto à suscetibilidade à meticilina e à presença do gene mecA. Destas, 49 (39.8%) foram identificados como MRSP. Os resultados confirmam a importância do monitoramento de patógenos resistentes e a necessidade de mais estudos para determinar a prevalência de MRSP em animais de companhia.

Characterization of methicillin-resistant Staphylococcus (MRS) is a continuous challenge at diagnostic laboratories. The phenotypic methods present heterogeneous results and the occurrence of variants of mecA gene turned this goal even more challenging to achieve. The present study provided an accurate and highly discriminatory screening tool for MRS, improving its detection.

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. RESUMO 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.

The present work evaluated the species distribution associated with the antimicrobial susceptibility pattern by phenotypic analysis and mec and bla gene detection in 100 Staphylococcus strains from 219 clinical samples from cats and dogs in the state of Rio de Janeiro, Brazil. Oxacillin susceptibility profiles were detected by the Clinical and Laboratory Standards Institute (CLSI) recommended tests. The amplification of the mecA gene was positive in 25% of the Staphylococcus spp. The whole mec complex (mecA-mecI-mecR1) was detected in four phenotypically oxacillin-resistant isolates. The CLSI recommended nitrocefin-based test detected 38% (38/100) β-lactamase producers Staphylococcus strains. Also, 32% (32/100) Staphylococcus spp. strains tested positive for bla genes. The whole bla gene complex, blaZ-blaI-blaR1, was detected in 7.8% (3/38) of the nitrocefinase-positive isolates. β-Lactamases was well spread among the samples and it seems to be a prevalent mechanism in resistant staphylococci strains from pet animals. The mec and bla gene regulatory systems can interfere in expression of resistance mediated by PBPs and β-lactamases, conferring the heterogeneous oxacillin-resistance in Staphylococcus spp. detected by phenotypic tests. Key words: Staphylococcus spp., oxacillin, β-lactamase, mecA, pet animals.

Staphylococcus aureus antimicrobial resistance, especially to beta-lactams, favors treatment failures and its persistence in herd environment. This work aimed to develop a more specific primer for mecA gene detection based on the comparison of the conserved regions from distinct host origins and also investigated the presence of homologue mecA(LGA251) in bovine strains. A total of 43 Staphylococcus spp. were included in this study, comprising 38 bovine S. aureus, two human and three equine coagulase-negative staphylococci (CNS). Phenotypical methicillin-resistance detection was performed through oxacillin agar-screening and cefoxitin disk-diffusion test. None isolate tested positive for mecA(LGA251) gene. For mecA gene PCR, new primers were designed based on the sequences of human S. aureus (HE681097) and bovine S. sciuri (AY820253) mecA. The new primers based on the S. aureus mecA sequence amplified fragments of human and equine CNS and the ones based on S. sciuri mecA sequence only yielded fragments for S. aureus bovine strains. Multiples alignments of mecA gene sequences from bovine, human and equine revealed punctual but significant differences in bovine strains that can lead to the mecA gene detection impairment. The observed divergences of mecA gene sequences are not a matter of animal or human origin, it is a specificity of bovine samples.

Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.