Effect of polycaprolactone nanocapsules loaded with essential oils on biofilm formation by Staphylococcus aureus strains isolated from bovine mastitis cases

Abstract Bovine infectious mastitis is largely resistant to antibacterial treatment, mainly due to mechanisms of bacterial resistance in the biofilms formed by Staphylococcus aureus. Melaleuca (MEO) and citronella essential oils (CEO) are promising agents for reducing or eliminating biofilms. Free melaleuca oil presented a medium Minimum Inhibitory Concentration (MIC) of 0.625% and a Minimum Bactericidal Concentration (MBC) of 1.250%, while free citronella oil showed medium MIC and MBC of 0.313%. Thus, free CEO and MEO demonstrate bacteriostatic and bactericidal potential. We generated polymeric nanocapsules containing MEO or CEO and evaluated their efficacy at reducing biofilms formed by S. aureus. Glass and polypropylene spheres were used as test surfaces. To compare the responses of free and encapsulated oils, strains were submitted to 10 different procedures, using free and nanoencapsulated essential oils (EOs) in vitro. We observed no biofilm reduction by MEO, free or nanoencapsulated. However, CEO nanocapsules reduced biofilm formation on glass (p=0.03) and showed a tendency to diminish biofilms on polypropylene (p=0.051). Despite nanoencapsulated CEO reducing biofilms in vitro, the formulation could be improved to modify the CEO component polarity and, including MEO, to obtain more interactions with surfaces and the biofilm matrix

Training, development, and evaluation of on-farm culturing as a technical resource to reduce the use of antimicrobials in dairy herds in the Midwest Region of Santa Catarina State, Brazil / Capacitação, implementação e avaliação da cultura de leite na fazenda como recurso técnico voltado ao uso reduzido de antimicrobianos em rebanhos leiteiros no meio oeste catarinense

ABSTRACT: On-farm culturing is a technology booming in Brazil. It is based on the microbiological diagnosis of milk samples collected from clinical cases of bovine mastitis, on the farm where the animals are handled, quickly, simply, and at an affordable cost. With this resource, after 24 h of incubation, the isolated microorganisms are classified as gram positive or gram negative, helping to make decisions regarding the therapy of the animals. Considering the relevance of the Midwestern region of Santa Catarina State in the national dairy production scenario, the objectives of the study were: 1) to promote the technical training of the farmers in three dairy farms located in Treze Tílias-SC, 2) to assist in the implementation and evaluation of on-farm culturing as technological resource viewing the reduced use of antimicrobials in herds. In addition to the rapid acceptance by the farmers and their efficient training, with the use of the technology, there was a 45-50% reduction in the use of antimicrobials in dairy herds. This result demonstrated a significant effect on animal health, and determines an important cost reduction for farmers, as presented in detail in this manuscript.

RESUMO: A cultura de leite na fazenda (do inglês On farm culturing) é uma tecnologia em franca expansão no Brasil. Fundamenta-se no diagnóstico microbiológico de amostras de leite colhidas de casos clínicos de mastite bovina, na própria fazenda onde são manejados os animais, de forma rápida, simples e com custo acessível. Com este recurso, após 24 horas de incubação, os micro-organismos isolados são classificados em Gram-positivos ou Gram-negativos, auxiliando na tomada de decisões quanto à terapia dos animais. Considerando a relevância da região meio oeste catarinense no cenário nacional de produção leiteira, o objetivo do presente estudo foi promover a capacitação técnica dos produtores em três propriedades localizadas em Treze Tílias-SC, e auxiliar na implementação e avaliação da cultura de leite na fazenda como recurso tecnológico voltado ao uso reduzido de antimicrobianos nos rebanhos. Além da rápida aceitação pelos produtores e eficiente capacitação dos mesmos, verificou-se, com o emprego da tecnologia, redução de 45% a 50% do uso de antimicrobianos nos rebanhos. Este resultado, além de demonstrar significativo impacto em saúde animal, determinou importante redução de custos aos produtores, como apresentado detalhadamente no presente artigo.

In vitro lytic efficiency of Staphylococcus aureus bacteriophages in bacteria from bovine mastitis: a meta-analysis / Eficiência lítica in vitro de bacteriófagos de Staphylococcus aureus em bactérias de mastites bovina: uma meta-análise

ABSTRACT: Bacteriophages have been investigated as alternative to the treatment of bacterial infections, including bovine mastitis, in production animals. In this meta-analysis, we evaluated in vitro efficiency of phages of Staphylococcus aureus against S. aureus, which is involved in the etiology of bovine mastitis. Seventeen studies were included and the bacterial lytic activity was extracted using proportion analysis. The lytic efficiency of phages was obtained in this meta-analysis using a random-effects model [significant difference (P<0.05)]. Forest plots were used to graphically represent the efficiency of phages on bacterial isolates. Most phages (e.g., CS1, DW2, ΦSA011, ΦSA012, ΦSA022, ΦSA023, ΦSA024, ΦSA025, ΦSA037, ΦSA038, ΦSA039, ΦSA041, ΦSA042, ΦSA043, ΦSA044, MSA6, Ufv-aur2 to Ufv-aur11, SAH-1, SPW, vB_SauM_JS25, SaPh1 to SaPh6, SA, SANF, SA2, ΦSA012, ΦSA039, phi11, phiIPLA88, phiIPLA35, phiIPLA-RODI, phiIPLA-C1C, SAJK-IND, vBSP-A1, vBSP-A2, STA1.ST29, EB1.ST11, EB1.ST27, Remus, and ISP) were efficiently lytics or infected most S. aureus isolates, demonstrating 80% (P<0.05) lytic efficiency. The phages SA, SANF and SA2, also demonstrated lytic activity or infected the non-Staphylococcus aureus and Macrococcus caseolyticus isolates. In this meta-analysis, we compared and demonstrated the in vitro efficiency and host range of S. aureus phages. Additionally, the phages represent an alternative to be researched to treat bovine mastitis in dairy cattle caused by the prevalent microorganism, S. aureus.

RESUMO: Os bacteriófagos têm sido investigados como alternativa ao tratamento de infecções bacterianas em animais de produção, incluindo a mastite bovina. Nesta meta-análise, avaliamos a eficiência in vitro de fagos de Staphylococcus aureus contra S. aureus envolvidas na etiologia da mastite bovina. Dezessete estudos foram incluídos e a atividade lítica bacteriana foi extraída usando análise de proporção. A eficiência lítica dos fagos foi obtida nesta meta-análise, usando um modelo de efeitos aleatórios (diferença significativa (P <0,05)). Os gráficos de Forest plots foram usados para representar graficamente a eficiência dos fagos em isolados bacterianos. Os fagos avaliados, na sua grande maioria, (por exemplo, CS1, DW2, ΦSA011, ΦSA012, ΦSA022, ΦSA023, ΦSA024, ΦSA025, ΦSA037, ΦSA038, ΦSA039, ΦSA041, ΦSA042, ΦSA043, ΦSAf e U0f, ua04 SPW, vB_SauM_JS25, SaPh1 a SaPh6, SA, SANF, SA2, ΦSA012, ΦSA039, phi11, phiIPLA88, phiIPLA35, phiIPLA-RODI, phiIPLA-C1C, SAJK-IND, vBSP-A1, vBSP-A2, STA1.ST29, EB1.ST11, EB1.ST27, Remus, e ISP) foram eficientemente líticos ou infectaram a maioria dos isolados de S. aureus, demonstrando 80% (P < 0,05) de eficiência lítica. Os fagos SA, SANF e SA2 também demonstraram atividade lítica ou infectaram os isolados Staphylococcus não-aureus e Macrococcus caseolyticus. Nesta meta-análise, comparamos e demonstramos a eficiência in vitro e gama de hospedeiros de fagos de S. aureus. Adicionalmente, os fagos representam uma alternativa a ser pesquisada para o tratamento da mastite bovina em gado leiteiro causada pelo microrganismo prevalente, ou seja S. aureus.

Virulence, agr groups, antimicrobial resistance and epidemiology of Staphylococcus aureus isolated from bovine subclinical mastitis / Virulência, grupos agr, resistência a antimicrobianos e epidemiologia de isolados de Staphylococcus aureus obtidos de vacas com mastite

Fifty-two Staphylococcus aureus recovered from papillary ostium and milk samples collected from cows with subclinical mastitis and milking environments in three small dairy herds located in southeastern Brazil were subjected to PCR identification based on the thermonuclease (nuc) gene. All the strains were submitted to in vitro antimicrobial susceptibility testing, and we investigated the sequence types (STs), agr groups (I-IV), virulence genes encoding for Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), biofilm-associated proteins, bi-component toxins, pyrogenic toxin superantigens, and enterotoxins. Screening for oxacillin resistance (2-6 µg/ml oxacillin), beta-lactamase activity assays, and PCR for the mecA/mecC genes detected 26 methicillin-susceptible S. aureus(MSSA) and 26 mec-independent oxacillin-nonsusceptible S. aureus (MIONSA). While MSSA isolates were found to be susceptible to all antimicrobial agents tested, or only resistant to penicillin and ampicillin, MIONSA isolates were multidrug-resistant. ST126-agr group II MSSA isolates were prevalent in milk (n=14) and carried a broad set of virulence genes (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla, and hlb), as well as the ST126-agr group II MIONSA isolated from milking liners (n=1), which also carried the eta gene. ST1-agr group III MIONSA isolates (n=4) were found in papillary ostium and milk, but most MIONSA isolates (n=21), which were identified in both papillary ostium and milking liners, were agr-negative and assigned to ST126. The agr-negative and agr group III lineages showed a low potential for virulence. Studies on the characterization of bovine-associated MSSA/MIONSA are essential to reduce S. aureus mastitis to prevent economic losses in dairy production and also to monitor the zoonotic potential of these pathogens associated with invasive infections and treatment failures in healthcare.

Cinquenta e dois isolados de Staphylococcus aureus obtidos de amostras colhidas do óstio papilar, do leite de vacas com mastite subclínica e do ambiente de ordenha em três fazendas de rebanhos leiteiros localizadas no sudeste do Brasil foram identificados por PCR para o gene da termonuclease (nuc). Todos os isolados foram testados para sensibilidade a antimicrobianos e foram investigados os sequence types (STs), grupos agr (I-IV) e genes de virulência que codificam Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), proteínas associadas a biofilme, toxinas bi-componentes, toxinas pirogênicas com propriedades de superantígenos e enterotoxinas. Triagem para detecção de resistência à oxacilina (2-6 µg/ml oxacilina), ensaios de atividade de enzimas beta-lactamases e PCR para os genes mecA/mecC detectaram 26 estirpes de S. aureus sensíveis à meticilina (methicillin-susceptible S. aureus, MSSA) e 26 estirpes de S. aureus mec-negativas não sensíveis à meticilina (mec-independent oxacillin-nonsusceptible S. aureus, MIONSA). Enquanto os isolados MSSA foram sensíveis a todos os agentes antimicrobianos testados, ou apenas resistentes à penicilina e ampicilina, os isolados MIONSA foram multirresistentes. MSSA ST126-agr grupo II foram prevalentes no leite (n= 14) e apresentaram um amplo conjunto de genes de virulência (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla e hlb), assim como o isolado MIONSA ST126-agr grupo II proveniente de um insuflador (n= 1), o qual também apresentou o gene eta. MIONSA ST1-agr grupo III (n= 4) foram identificados no óstio papilar e leite, mas a maioria dos isolados MIONSA (n= 21), encontrados em óstios papilares e insufladores, foram agr-negativos e pertenceram ao ST126. As linhagens agr-negativas e agr grupo III apresentaram baixo potencial de virulência. Estudos sobre a caracterização de MSSA/MIONSA associados a bovinos são essenciais para a redução da mastite causada por S. aureus e de perdas econômicas na produção leiteira e, também, para o monitoramento do potencial zoonótico desses patógenos associados a infecções invasivas e falhas de tratamento em ambientes hospitalares.

Changes in somatic cell count, composition and cytokine levels in milk from cows with mastitis due to mixed infections

Mastitis is a mammary gland inflammation that is very common worldwide, mostly caused by bacteria, and causes enormous economic losses. Many microorganisms cause this disease. The most common causes of mastitis by these microorganisms are Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Streptococcus agalactiae (S. agalactiae). The anti-inflammatory properties of transforming growth factor (TGF)-ß include: 1) limiting interferon (IFN)-γ production; 2) increasing the expression of the interleukine (IL)-1 receptor antagonist; 3) inhibiting macrophage production of chemokines, pro-inflammatory cytokines, nitric oxide, and reactive oxygen intermediates; and 4) increasing macrophage clearance of bacterial debris and damaged parenchymal cells. It is stated that cytokines and milk composition change in case of mastitis. In this study, it was aimed to reveal the changes in milk TGF-ß1 and Tumor necrosis factor (TNF)-α concentrations and milk composition in mixed infections caused by three pathogens causing mastitis. In this study, milk samples from 90 cows were divided into 5 groups. Tumor necrosis factor (TNF)-α and TGF-ß1 concentrations and milk composition were determined in these milk samples. The California Mastitis Test (CMT) was applied to the cows included in the study and scoring was done. According to the CMT results of the milk samples taken, CMT(-) cows were included in group 1 (n = 22). Those with the CMT(+) were sent to the microbiology laboratory for analysis within 2 h. After the bacteria was determined, combination groupings were formed. Group 2 (n = 17), in which S. aureus and E. coli grew together, group 3 (n = 21), in which S. aureus and S. agalactiae grew together, group 4 (n = 8), in which S. agalactiae and E. coli grew together in milk samples, and milk samples without any bacterial growth in CMT (+) formed group 5 (n = 22), respectively. Somatic cell count was measured with the DeLaval Cell Counter® (Cell Counter DCC) device. Mineral matter, fat, protein, lactose, electrical conductivity and specific gravity were measured in milk samples using Lactoscan Milk Analyzer (Milkotronic/EUROPE). Milk samples were then stored at -80°C to measure TGF-ß1 and TNF-α. Tumor necrosis factor-α and TGF-ß1 concentrations in milk samples were measured using ELISA kits (Sunred Biological Technology). Changes in milk TNF-α and TGF-ß1 concentration and milk composition were determined in milk samples with mastitis caused by mixed infection. The TNF-α concentration of group 4 was higher than the other groups. On the other hand, the highest concentration of TGF-ß1 was found in group 2. While the number of somatic cells in group 1 was lower than in groups 2, 3, and 4, there was no statistical difference between groups 1 and 5. The lowest milk fat ratio was found in group 1, and it was found to be statistically lower than groups 2, 3, and 4. While the rate of solid-non-fat of group 1 increased compared to groups 2 and 3, the highest protein ratio was found in groups 1 and 5. There was no difference between the 5 groups in terms of mineral matter ratios. While the specific gravity was highest in group 1, there was no statistical difference between the other 4 groups. Overall, it was concluded that there was an increase in TNF-α and TGF-ß1 concentrations and a change in milk composition in samples with bacterial growth.(AU)

Detection of icaA, icaD, and bap genes and biofilm production in Staphylococcus aureus and non-aureus staphylococci isolated from subclinical and clinical bovine mastitis / Detecção dos genes icaA, icaD e bap e produção de biofilme por Staphylococcus aureus e estafilococos não-aureus isolados de mastite clínica e subclínica bovina

Algumas espécies de Staphylococcus causam infecções crônicas intramamárias e podem levar à formação de biofilme. No presente estudo, levantou-se a hipótese de que as espécies de Staphylococcus isolados da mastite bovina são capazes de formar biofilme in vitro associado à presença dos genes icaA, icaD ou bap. Um total de 200 isolados de Staphylococcus, sendo 100 Staphylococcus aureus de casos de mastite subclínica e 100 estafilococos não aureus (ENA) de casos de mastite subclínica e clínica, obtidos em duas fazendas leiteiras, no estado de São Paulo, foram avaliados quanto à capacidade de produzir biofilmes in vitro. A presença de icaA, icaD e bap foi confirmada por PCR, e a produção de biofilme em ágar vermelho congo (Congo Red Agar - CRA) e em teste de microplaca (Microtiter Plate - MtP) foi avaliada nos isolados de S. aureus e ENA. Os resultados mostraram a presença dos genes icaA, icaD e bap em S. aureus, mas não em ENA. A produção de biofilme pode estar associada à presença de outros fatores ou genes que estimulam a produção de biofilme in vitro. O ensaio de MtP serve como um modelo quantitativo para o estudo da aderência de espécies de estafilococos associados à mastite bovina.(AU)

Microbiological and molecular detection of Mycoplasma bovis in milk samples from bovine clinical mastitis / Detecção microbiológica e molecular de Mycoplasma bovis em amostras de leite oriundas de mastite clínica bovina

The genus Mycoplasma includes more than 200 bacterial species that cause disease in animals. It is responsible for causing mastitis in bovines and may be related to other manifestations, such as arthritis and pneumonia in calves and heifers. The present study aimed to detect Mycoplasma bovis isolated from milk samples of bovine clinical mastitis, and to compare the isolation rates in two culture media: Hayflick and SP4. An initial screening was performed in order to detect the presence of the class Mollicutes in 1166 milk samples from clinical mastitis by the conventional Polymerase Chain Reaction (PCR) technique. According to the 1166 milk samples evaluated, 8.6% (100/1166) were positive to class Mollicutes. Regarding molecular analyses, 1.1% (13/1166) of conventional PCR for positive M. bovis was obtained and 0.9% (11/1166) in real-time PCR. The results of the microbiological culture of the 100 samples previously screened demonstrated that 6% (6/100) of colony growth have been developed when using the Hayflick medium, and 11% (11/100) when using the SP4 medium (including the positive on Hayflick medium). Concerning the 11 isolates obtained in the microbiological culture, conventional PCR confirmed M. bovis in nine of them, and two cultures were negative. In the phylogenetic analysis of the isolates, all of them were grouped in M. bovis and M. agalactiae clusters. The results confirmed the importance of the presence of M. bovis in the etiology of bovine clinical mastitis and reinforced the need for further studies to elucidate other Mycoplasma species that may be involved in bovine clinical mastitis in Brazil.(AU)

O gênero Mycoplasma inclui mais de 200 espécies que causam doenças nos animais. É responsável por quadros de mastite em bovinos, podendo também estar relacionado à outras manifestações como artrite e pneumonia em bezerros e novilhas. O presente estudo objetivou a detecção de Mycoplasma bovis isolados a partir de amostras de leite de mastite clínica bovina, bem como, a comparação da taxa de isolamento em dois meios de cultura: Hayflick e SP4. Para efeito de triagem amostral, foram avaliadas quanto à presença da classe Mollicutes 1166 amostras de leite de casos de mastite clínica pela técnica de PCR convencional. Das 1166 amostras de leite avaliadas, 8,6% (100/1166) foram positivas à classe. Nas análises moleculares, obteve-se 1,1% (13/1166) de positividade para Mycoplasma bovis na PCR convencional e 0,9% (11/1166) na PCR em tempo real. Os resultados do cultivo microbiológico das 100 amostras triadas previamente demonstraram 6% (6/100) de crescimento de colônias ao se utilizar o meio Hayflick e 11% (11/100) ao se utilizar o meio SP4 (incluindo as positivas ao primeiro). A partir dos 11 isolados obtidos no cultivo microbiológico, a PCR convencional confirmou Mycoplasma bovis em nove deles, e dois foram negativos para o agente. Na análise filogenética dos isolados, todos agruparam no cluster Mycoplasma bovis e Mycoplasma agalactiae. Frente aos resultados, ressalta-se a importância da presença de Mycoplasma bovis na etiologia da mastite clínica bovina e reforça a necessidade de estudos mais aprofundados para a elucidação de outras espécies de micoplasmas que possam estar envolvidas na mastite bovina.(AU)

Seasonal variation of propolis from southern Brazil: phytochemical screening, antimicrobial activity, and effects on bovine mammary epithelial cells / Variação sazonal da própolis do Sul do Brasil: screening fitoquímico, atividade antimicrobiana e efeitos em células epiteliais mamária bovina

Previous studies have reported on the phytotherapeutic potential of propolis from southern Brazil (Urupema, Santa Catarina State), in particular, its efficacy in the treatment of bovine mastitis. The present study evaluates the effect of seasonal variation on the chemical composition of propolis from southern Brazil and its resultant antimicrobial and cytotoxic activities in the context of treating bovine mastitis. Antimicrobial activity against Staphylococcus aureus was assessed, along with the cytotoxicity and induction of apoptosis in MAC-T bovine mammary epithelial cells. With the exception of spring propolis, the flavonoid quercetin was the main compound present in all samples. The minimum inhibitory concentration (MIC) of propolis against S. aureus from mastitic milk was 140 µg/mL for samples collected during the spring, autumn, and winter seasons, but 280 µg/mL for samples collected in summer. For MAC-T cells, the spring propolis extract was more toxic with an IC50 of 120 µg/mL. However, for 120 µg/mL of spring propolis extract, only 0.77% of necrotic and 37% of apoptotic MAC-T cells were found, respectively. Nonetheless, the induction of cell death through apoptosis by propolis extract suggests less severe damage to bovine mammary glands. Moreover, only negligible seasonal variation was found in the chemical composition of propolis from southern Brazil, and no biological activities were determined to be harmful. Therefore, this propolis shows promise as an alternative to commercial antimicrobials in the control of bovine mastitis, offering support for organic milk production.(AU)

Estudos prévios tem demonstrado o potencial terapêutico da propolis do Sul do Brasil (Urupema, Santa Catarina), em particular no tratamento da mastite bovina. O presente estudo tem como objetivo avaliar o efeito da variação sazonal sobre a composição química da própolis de Urupema do Sul do Brasil e suas atividades antimicrobiana e citotóxica visando o tratamento da mastite bovina. A atividade antimicrobiana contra Staphylococcus aureus foi avaliada, juntamente com a citotoxicidade e indução de apoptose em células epiteliais mamárias bovina da linhagem MAC-T. Com exceção da própolis da primavera, o flavonóide quercetina foi o composto majoritário em todas as amostras. A concentração inibitória mínima (CIM) da propolis contra S. aureus de leite mastítico foi 140 µg/mL para as amostras de primavera, outono e inverno e 280 µg/mL para a amostra coletada no verão. Para as células MAC-T, o extrato de própolis da primavera foi o mais tóxico, sendo a IC50 120 µg/mL. Entretanto, com 120 µg/mL do extrato de própolis primaveril, somente 0,77% de células MAC-T necróticas e 37% apoptóticas foram encontradas. Portanto, a indução da morte celular por apoptose pelo extrato de própolis sugere danos possivelmente menos graves a glândula mamária bovina. Além disso, somente uma pequena variação sazonal foi encontrada para a composição química da propolis do Sul do Brasil, a qual não prejudicou suas atividades biológicas. Portanto, esta própolis mostra como uma alternativa promissora ao uso de antimicrobianos comerciais no controle da mastite bovina e uma opção para subsidiar a produção orgânica de leite.Palavras-chave: Células MAC-T. Mastite bovina. Propolis brasileira. Staphylococcus aureus.(AU)

Biofilm formation by Prototheca zopfii isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes / Formação de biofilme por Prototheca zopfii isolados de mastite bovina clínica e subclínica em distintas condições de crescimento sob diferentes corantes

ABSTRACT: Prototheca spp. have been reported as an emergent environmental mastitis pathogen in several countries. Biofilm formation is a significant factor associated with different degrees of virulence developed by many microorganisms, including Prototheca spp. The present study aimed to compare two growth conditions and two staining dyes to determine which combination was more appropriate to evaluate qualitatively and quantitatively the production of biofilm by P. zopfii. Biofilm formation was evaluated in polystyrene microplates under static and dynamic growth conditions and staining with crystal violet or cotton blue dye. All P. zopfii isolates from cows with mastitis were classified as biofilm-producers in all growth conditions and staining. The cotton blue dye proved to be more appropriate method to classify the intensity of P. zopfii biofilm production.

RESUMO: Prototheca spp. tem sido relatado como um patógeno ambiental causador de mastite bovina em vários países. A formação de biofilme é um fator associado a diferentes graus de virulência desenvolvidos por muitos microrganismos, incluindo Prototheca zopfii. O presente estudo teve como objetivo comparar duas condições de crescimento e dois corantes para determinar a combinação mais adequada para avaliar qualitativa e quantitativamente a produção de biofilme por P. zopfii. A formação de biofilme foi avaliada em microplacas de poliestireno sob condições estáticas e dinâmicas de crescimento e coloração com cristal violeta ou azul de algodão. Todos os isolados de P. zopfii de vacas com mastite foram caracterizados como produtores de biofilme, independentemente das condições de crescimento e coloração. O corante azul de algodão demonstrou ser o método mais adequado para classificar a intensidade de produção de biofilme de P. zopfii.

Identification and antimicrobial suceptibility profile of bacteria causing bovine mastitis from dairy farms in Pelotas, Rio Grande do Sul / Identificação e perfil de suscetibilidade antimicrobiana de bactérias causadoras de mastite bovina em propriedades leiteiras de Pelotas, Rio Grande do Sul

Abstract Mastitis is an inflammatory process of the udder tissue caused mainly by the bacteria Staphylococcus aureus. The indiscriminate use of antibiotics fosters conditions that favor the selection of resistant microorganisms, suppressing at the same time susceptible forms, causing a serious problem in dairy cattle. Given the importance in performing an antibiogram to select the most adequate antimicrobial therapy, the aim of this study was to identify bacteria isolated from cow's milk with mastitis, in dairy farms situated in the city of Pelotas, Rio Grande do Sul, and to determinate the susceptibility profile of these isolates against the antibiotics used to treat this illness. A total of 30 isolates of Staphylococcus spp., were selected from milk samples from the udder quarters with subclinical mastitis whose species were identified through the Vitek system. The susceptibility profile was performed by the disk diffusion assay, against: ampicillin, amoxicillin, bacitracin, cephalexin, ceftiofur, enrofloxacin, gentamicin, neomycin, norfloxacin, penicillin G, tetracycline and trimethoprim. In the antibiogram, 100.0% of the isolates were resistant to trimethoprim and 96.7% to tetracycline and neomycin, three strains of Staphylococcus spp., (10.0%) presented resistance to the 12 antibiotics tested and 24 (80.0%) to at least eight. These results showed the difficulty in treating mastitis, due to the pathogens' resistance.

Resumo A mastite se constitui no processo inflamatório da glândula mamária causada principalmente por bactérias Staphylococcus aureus. O uso indiscriminado dos antibióticos promove condições que favorecem a seleção de micro-organismos resistentes e, ao mesmo tempo, suprime formas suscetíveis, causando um grave problema para a bovinocultura leiteira. Tendo em vista a importância da realização do antibiograma para a seleção da terapia antimicrobiana mais adequada, o objetivo deste estudo foi identificar bactérias isoladas de leite de vaca com mastite, oriundas de propriedades leiteiras localizadas na cidade de Pelotas, RS, bem como determinar o perfil de suscetibilidade desses isolados frente a antibióticos usados para o tratamento desta doença. Foram selecionados 30 isolados de Staphylococcus spp. de amostras de leite provenientes de quartos mamários com mastite subclínica, cujas espécies foram identificadas através do sistema Vitek. O perfil de suscetibilidade foi realizado pela técnica de difusão em disco, frente a: ampicilina, amoxicilina, bacitracina, cefalexina, ceftiofur, enrofloxacina, gentamicina, neomicina, norfloxacina, penicilina G, tetraciclina e trimetoprima. No antibiograma, 100,0% dos isolados foram resistentes a trimetoprima e 96,7% a tetraciclina e a neomicina, três cepas (10,0%) foram resistentes aos 12 antibióticos testados e 24 (80,0%) a pelo menos oito. Esses resultados demonstram a dificuldade encontrada no tratamento da mastite devido à resistência dos agentes patológicos.

Rapid identification of bovine mastitis pathogens by MALDI-TOF Mass Spectrometry / Rápida identificação de agentes causadores de mastite por espectrometria de massas MALDI-TOF

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to be an alternative method for identification of bacteria via their protein profile spectra, being able to identify bacteria at the genus, species and even at subspecies level. With the aim of large-scale identification of pathogens causing mastitis by this platform, a total of 305 isolates of bacteria identified from cows with subclinical mastitis were analyzed by conventional microbiological culture (MC) as well as by MALDI-TOF MS coupled with Biotyper data processing. Approximately 89% of the identifications performed by MALDI-TOF MS were consistent with results obtained by MC. From the remaining isolates (11%), 6.3% of isolates were classified as misidentified (discordance for both genus and species level), and 4.7% showed identification agreement at the genus level but not at the species level, being classified as unidentified at species level. The disagreement results were mostly associated with identification of Streptococcus and Enterococcus species probably due to the narrow phenotypic similarity between these two genera. These disagreement results suggest that biochemical assays might be prone to identification errors and, MALDI-TOF MS therefore may be an alternative to overcome incorrect species-specific identification. Standard microbiological methods for bovine mastitis diagnosis are time consuming, laborious and prone to errors for some bacteria genera. In our study, we showed that MALDI-TOF MS coupled with Biotyper may be an alternative method for large-scale identification of bacteria isolated from milk samples compared to classical microbiological routine protocols.(AU)

A espectrometria de massas (MALDI-TOF MS) tem mostrado ser um método alternativo para a identificação de bactérias, sendo capaz de identificar as bactérias causadoras de mastite em gênero, espécie ou até mesmo subespécie. Com o objetivo de identificar os patógenos causadores de mastite em grande-escala por esta plataforma, um total de 305 isolados bacterianos oriundos de vacas com mastite subclínica foram analisados pela cultura microbiológica convencional (CM) e pela MALDI-TOF MS acoplada ao software Biotyper. Aproximadamente 89% das identificações realizadas pela MALDI-TOF MS foram consistentes com os resultados obtidos pela CM. Do restante de isolados bacterianos (11%), 6,3% foram classificados como identificação errônea (discordância de gênero e espécie), e 4,7% apresentaram concordância de gênero, mas discordância da espécie. Os resultados que apresentaram divergência estavam mais associados com a identificação das espécies de Streptococcus spp. e Enterococcus spp. devido à similaridade fenotípica entre os dois gêneros. Estes resultados divergentes sugerem que os ensaios bioquímicos podem ser propensos a erros de identificação, por isso a MALDI-TOF MS pode ser considerada um método alternativo para superar os erros de identificação da CM. A cultura microbiológica padrão e os ensaios bioquímicos utilizados na identificação de agentes causadores de mastite são demorados, trabalhosos e propensos a erros quando utilizados na identificação em nível de espécie. No presente estudo, demonstramos que a MALDI-TOF MS acoplada ao software Biotyper pode ser considerada um método alternativo de identificação de bactérias causadoras de mastite em grande-escala quando comparado com a cultura microbiológica convencional.(AU)

Molecular prophage typing of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.

Molecular identification of coagulase-negative staphylococci by rpoB sequence typing / 대한수의학회지

Bovine mastitis (BM) has resulted in enormous economic loss in the dairy industry and coagulase-negative staphylococci (CNS) have caused subclinical BM. Although VITEK 2 GP ID card (VITEK 2) has been used for CNS identification, the probability of identification varies. The rpoB sequence typing (RSTing) method has been used for molecular diagnosis and epidemiology of bacterial infections. In this study, we undertook RSTing of CNS and compared the results with those of VITEK2 and 16S rRNA gene sequencing. As compared VITEK2, the molecular-based methods were more reliable for species identification; moreover, RSTing provided more molecular epidemiological information than that from 16S rRNA gene sequencing.

Produção de biofilme por Staphylococcus chromogenes isolados de amostras de leite provenientes de rebanhos bovinos com mastite / Biofilme production by Staphylococcus chromogenes isolated from milk samples from bovine with mastitis

Foram estudadas 135 vacas mestiças, provenientes de 10 rebanhos leiteiros no estado do Acre. O objetivo foi identificar espécies de Staphylococcus isoladas dos quartos mamários de vacas com mastite e, posteriormente, avaliar a capacidade de produção de biofilme pela espécie Staphylococcus chromogenes. A caracterização dos isolados presentes nas amostras encontradas, correspondentes a Staphylococcus sp., foi realizada utilizando-se a técnica do MALDI TOF MS (Matrix Associated Laser Desorption-Ionization - Time of Flight - Mass Spectrometry). Foram identificados: S. chromogenes (36), Staphylococcus saprophyticus (5), S. chromogenes ou Staphylococcus hycus (5), Staphylococcus haemolyticus (4), Staphylococcus epidermidis (3), Staphylococcus hycus (3), Staphylococcus aureus (1), Staphylococcus auriculares (1), Staphylococcus kloosii (1) e Staphylococcus xylosus (1). A espécie S. chromogenes correspondeu a 60% dos isolados do gênero (17 isolados coagularam o plasma de coelho no teste da coagulase em tubo), sendo 83,3% dos isolados (30/36) produtores de biofilme, não estando esse fator de virulência associado ao fenótipo de coagulação do plasma. A identificação desses microrganismos é importante para a elucidação da etiologia da mastite bovina. O alto percentual de S. chromogenes, produtores de biofilme, isolados de vacas com mastite é um achado relevante e pode revelar uma mudança de perfil na colonização de agentes etiológicos causadores dessa enfermidade.(AU)

A total of 135 crossbred cows was studied, from ten dairy herds on the state of Acre. The purpose was to identify species of Staphylococcus isolated from the mammary quarters of cows with mastitis. Additionally, the capacity of biofilm production by the species Staphylococcus chromogenes was evaluated. The sample characterization of the isolates was performed using the MALDI TOF MS (Matrix Associated Laser Desorption-Ionization - Time of Flight - Mass Spectrometry). The following species were identified by MALDI TOFF MS: S. chromogenes (36), Staphylococcus saprophyticus (5), S. chromogenes or Staphylococcus hycus (5), Staphylococcus haemolyticus (4), Staphylococcus epidermidis (3), Staphylococcus hycus (3), Staphylococcus aureus (1), Staphylococcus auriculares (1), Staphylococcus kloosii (1) and Staphylococcus xylosus (1). S. chromogenes corresponded to 60% of the Staphylococcus isolates (17 isolates positive on tube coagulase test). From those, 83.33% (30/36) of them were biofilm producers. This virulence factor had no association with the plasma coagulation phenotype. The identification of these microorganisms is important for the elucidation of the bovine mastitis etiology. The high percentage of S. chromogenes, biofilm producers, isolated from cows with mastitis is an important finding. This may reveal a profile change on the colonization of etiologic agents that cause this disease.(AU)

Considerações sobre o tratamento das mastites / Considerations on the treatment of mastitis

A mastite é considerada o maior problema dos animais destinados à produção de leite. Altera a sua composição e aumenta a contagem de células somáticas (CCS). Os micro-organismos envolvidos na doença podem ser origem infecciosa, como Staphylococcus aureus, ou ambiental, tal como Escherichia coli. A cultura bacteriana é uma ferramenta de diagnóstico e auxilia na detecção do patógeno causador da mastite. No entanto, fatores como fagocitose podem desencadear um resultado negativo. Quando estabelecido um programa de controle de mastite, o diagnóstico precoce e o início do tratamento adequado dos casos clínicos são fundamentais para se atingir os objetivos e seu sucesso, está relacionado com o patógeno envolvido. A indicação do tratamento de longa duração, ou terapia estendida, tem melhorado a resposta ao tratamento em casos de mastite por S. aureus, no entanto, com 30-50% de cura. Do ponto de vista do manejo dos animais, devido a alta contagiosidade deste patógeno, sua persistência no rebanho e custo em função ao tratamento, muitas vezes, o descarte do animal tem sido priorizado a fim de controlar os casos de mastite em propriedades. As medidas de controle são muito importantes para contribuir com a redução de casos de mastite por este patógeno. A indicação do tratamento intramamário associada com sistêmico tem poder efetivo em casos de mastite por E. coli, cujos casos agudos apresentam-se com sepse e toxemia. São abordados ainda aspectos de tratamentos alternativos das mastites, utilizados principalmente no processo orgânico de produção leiteira.(AU)

Mastites are considered a major problem on animals for milk production. Changes the milk composition and increases the somatic cell count (SCC). The microorganism involved in the disease may be infectious origin such as Staphylococcus aureus or environmental such as Escherichia coli. Bacterial culture is a diagnostic tool and aids in the detection of pathogenic causing masitis. However factor such as phagocytosis may result a negative result. When established a mastitis control program early diagnose and the initiation of appropriate treatment of clinical cases are fundamental for achieving the goals and success is related to the pathogen involved. The indication of treatment of long duration, or extended therapy has improved the response to the treatment in cases of matitis by S. aureus, however with 30-50% of cure. From the view point of handling of animals, given the high infectiousness of this pathogen, its persistence in the herd and cost-effective as a function of response to treatment often has prioritized the animal's discard in order to control the mastitis cases in properties. Control measures are very important to contribute the reduction of cases of mastitis symptoms by this pathogen. The indication of intramammary treatment associated with systemic has power effective in cases of mastitis by ­E. coli, whose acute cases present with sepsis and toxemia. Also address aspects of alternative treatments of mastitis, mainly used in the organic process of milk production.(AU)

The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment

Abstract Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n = 47) and fecal samples (n = 94) from cows, and samples from water (n = 23) and milk lines (n = 19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.

Identification and antimicrobial suceptibility profile of bacteria causing bovine mastitis from dairy farms in Pelotas, Rio Grande do Sul

Abstract Mastitis is an inflammatory process of the udder tissue caused mainly by the bacteria Staphylococcus aureus. The indiscriminate use of antibiotics fosters conditions that favor the selection of resistant microorganisms, suppressing at the same time susceptible forms, causing a serious problem in dairy cattle. Given the importance in performing an antibiogram to select the most adequate antimicrobial therapy, the aim of this study was to identify bacteria isolated from cows milk with mastitis, in dairy farms situated in the city of Pelotas, Rio Grande do Sul, and to determinate the susceptibility profile of these isolates against the antibiotics used to treat this illness. A total of 30 isolates of Staphylococcus spp., were selected from milk samples from the udder quarters with subclinical mastitis whose species were identified through the Vitek system. The susceptibility profile was performed by the disk diffusion assay, against: ampicillin, amoxicillin, bacitracin, cephalexin, ceftiofur, enrofloxacin, gentamicin, neomycin, norfloxacin, penicillin G, tetracycline and trimethoprim. In the antibiogram, 100.0% of the isolates were resistant to trimethoprim and 96.7% to tetracycline and neomycin, three strains of Staphylococcus spp., (10.0%) presented resistance to the 12 antibiotics tested and 24 (80.0%) to at least eight. These results showed the difficulty in treating mastitis, due to the pathogens resistance.

Resumo A mastite se constitui no processo inflamatório da glândula mamária causada principalmente por bactérias Staphylococcus aureus. O uso indiscriminado dos antibióticos promove condições que favorecem a seleção de micro-organismos resistentes e, ao mesmo tempo, suprime formas suscetíveis, causando um grave problema para a bovinocultura leiteira. Tendo em vista a importância da realização do antibiograma para a seleção da terapia antimicrobiana mais adequada, o objetivo deste estudo foi identificar bactérias isoladas de leite de vaca com mastite, oriundas de propriedades leiteiras localizadas na cidade de Pelotas, RS, bem como determinar o perfil de suscetibilidade desses isolados frente a antibióticos usados para o tratamento desta doença. Foram selecionados 30 isolados de Staphylococcus spp. de amostras de leite provenientes de quartos mamários com mastite subclínica, cujas espécies foram identificadas através do sistema Vitek. O perfil de suscetibilidade foi realizado pela técnica de difusão em disco, frente a: ampicilina, amoxicilina, bacitracina, cefalexina, ceftiofur, enrofloxacina, gentamicina, neomicina, norfloxacina, penicilina G, tetraciclina e trimetoprima. No antibiograma, 100,0% dos isolados foram resistentes a trimetoprima e 96,7% a tetraciclina e a neomicina, três cepas (10,0%) foram resistentes aos 12 antibióticos testados e 24 (80,0%) a pelo menos oito. Esses resultados demonstram a dificuldade encontrada no tratamento da mastite devido à resistência dos agentes patológicos.

A time series analysis of bulk tank somatic cell counts of dairy herds located in Brazil and the United States / Uma análise de séries temporais de contagem de células somáticas de tanque de rebanhos leiteiros localizados no Brasil e nos Estados Unidos

ABSTRACT: Bulk tank somatic cell counts (BTSCC) is widely used to monitore the mammary gland health at the herd and regional level. The BTSCC time series from specific regions or countries can be used to compare the mammary gland health and estimate the trend of subclinical mastitis at the regional level. Three time series of BTSCC from dairy herds located in the USA and the Southeastern Brazil were evaluated from 1995 to 2014. Descriptive statistics and a linear regression model were used to evaluate the data of the BTSCC time series. The mean of annual geometric mean of BTSCC (AGM) and the percentage of dairy herds with a BTSCC greater than 400,000 cells mL-1 (%>400) were significantly different (P<0.05) according to the countries and the times series. Linear regression model used for the USA time series was statistically significant for AGM and the %>400 (P<0.05). The first and second USA time series presented an increasing and decreasing trend for AGM and the %>400, respectively. The linear regression model for the Brazil time series was not significant (P>0.05) for both dependent variables (AGM and %>400). The Brazil time series showed no increasing or decreasing trend for the AGM and %>400. Consequently, approximately 40 to 50% of the dairy herds from southeastern Brazil will not achieve the regulatory limits for BTSCC over the next years.

RESUMO: Contagens de células somáticas de tanque (BTSCC) são amplamente utilizadas para monitorar a saúde da glândula mamária em níveis regional e de rebanho. Séries temporais de BTSCC, de regiões ou países específicos, podem ser usadas para comparar a saúde da glândula mamária e estimar a tendência de mastite subclínica em nível regional. Três séries temporais de BTSCC de rebanhos leiteiros, localizados nos EUA e na região sudeste do Brasil, foram avaliadas de 1995 a 2014. A estatística descritiva e modelos de regressão linear foram usados para avaliar as informações das séries temporais de BTSCC. A média anual geométrica de BTSCC (AGM) e a porcentagem de rebanhos com BTSCC, maior que 400.000 células mL-1 (%>400), foram significantemente diferentes (P<0,05) de acordo com os países e as séries temporais analisadas. O modelo de regressão linear usado para as séries temporais dos EUA foram estatisticamente significantes para AGM e %>400 (P<0,05). A primeira e a segunda séries temporais dos EUA apresentaram uma tendência de aumento e diminuição, tanto para AGM quanto para %>400, respectivamente. O modelo de regressão linear para as séries temporais do Brasil não foi significante (P>0,05) para ambas as variáveis dependentes (AGM e %>400). As séries temporais do Brasil não apresentaram tendência de aumento ou diminuição para AGM e %>400. Consequentemente, aproximadamente, 40 a 50% dos rebanhos leiteiros do sudeste do Brasil não alcançarão os limites regulatórios para BTSCC ao longo dos próximos anos.

Antibiotic activity of Plectranthus ornatus Codd, a Traditional Medicinal Plant

ABSTRACT The dichloromethane extract of Plectranthus ornatus Codd., a tradicional medicinal plant, showed antibiotic activity with minimum inhibitory concentration (MIC) values of 0.4 mg.mL-1 and 100 percent of biofilm inhibition against Staphylococcus aureus strains isolated from animals with mastitis infections. Based on these antibacterial activities, in addition to ethnopharmacological reports from healing men and farmers in Brazil, an herbal soap was produced from this active extract and was tested both in vitro and in vivo. In vivo assays conducted on these herbal soaps led to results similar to those previously conducted with the active extract. These results indicated the great potential of this plant for use as an excipient by preparing herbal antibacterial soaps as an alternative veterinary medicine aimed at controlling bovine mastitis infections on small Brazilian farms.

Identification of microbiome with 16S rRNA gene pyrosequencing and antimicrobial effect of egg white in bovine mastitis

Bovine mastitis is an important microbial disease in the dairy industry. We investigated the frequencies of bacterial pathogens in 62 farms and pathogen antibiotic resistance from mastitis samples (n = 748). We tested the antimicrobial activity of chicken and duck egg white and lysozyme purified from chicken egg white. Moreover, we compared the microbiomes of normal and mastitic raw milk obtained by 16S rRNA gene pyrosequencing and culture methods. The results showed that the frequencies of Gram-positive pathogens (Enterococcus faecalis 37% and Staphylococcus aureus 36%) were higher than that of a Gram-negative pathogen (Escherichia coli 15%). Resistance frequencies to ampicillin and norfloxacin were lowest in Staphylococcus aureus (21%), Enterococcus faecalis (23%), and Escherichia coli (33%), and the antimicrobial activity of chicken egg white was higher than those of lysozyme and duck egg white. Pyrosequencing results revealed clear differences between the microbiomes of mastitic and normal raw milk samples and revealed a slightly similar, but clearly different, composition of pathogens compared to that from the culture method. Thus, pyrosequencing may be useful for elucidating changes in microbiomes during mastitis progression and treatment. A chicken egg white and antibiotic combination may help with mastitis treatment; however, further studies are needed.