Resumo
An experiment was carried out to study the effect of different eubiotics on productive characteristics, intestinal integrity, as well as the content of enterobacteria in the cecum of broiler chickens. A completely randomized design with five treatments and 8 replicates of 25 birds each was used. In total 1000 mixed broiler chickens from Ross308 strain, one day old were obtained from a commercial hatchery. The birds were housed on concrete floors in a conventional house. A sorghum+soybean meal control diet was used, to which the additives under study were added. The treatments were distributed as follows: T1 = Control diet without antibiotic or eubiotic; T2 = T1 + bacteriophages; T3 = T1 + antibiotic; T4 = T1 + probiotic; T5 = T1 + symbiotic. The results obtained at 49 days of age for weight gain and feed conversion rate improved (p<0.05) with the addition of the antibiotic and eubiotics. A lower (p<0.05) intestinal density was observed with the probiotic. The height, width, and area of villi in duodenum was higher (p<0.05) when antibiotic and eubiotics were included. In the histological score, in duodenum, the antibiotic and eubiotics resulted with a higher score (p<0.05), associated to a physiological and controlled inflammation response that allowed improving productivity. Finally, the relative expression of enterobacteria, such as Lactobacillus salivarius, allowed associating positive changes in the microbiome and better productive parameters when including the symbiotic, with comparable results to the antibiotic when including the eubiotics.(AU)
Assuntos
Animais , Galinhas/microbiologia , Infecções por Enterobacteriaceae/diagnóstico , Enterobacteriaceae , Aditivos Alimentares/análise , Fenômenos Fisiológicos da Nutrição AnimalResumo
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.
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.
Assuntos
Feminino , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Mastite Bovina/microbiologiaResumo
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.
Resumo
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.(AU)
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.(AU)
Assuntos
Animais , Feminino , Bovinos , Doenças dos Bovinos/diagnóstico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Mastite Bovina/microbiologiaResumo
Os bacteriófagos ou fagos são vírus que possuem a capacidade de infectar e destruir as bactérias, por meio da lise celular. Esses vírus podem representar uma alternativa promissora para o tratamento das infecções bacterianas, especialmente àquelas causadas por agentes mutirresistentes às terapias antimicrobianas, como as mastites bovinas por Staphylococcus aureus, e no controle de biofilmes bacterianos. Sendo assim, os objetivos deste estudo foram: isolar e caracterizar fagos de S. aureus provenientes de leite bovino; testar a eficiência dos fagos frente aos isolados bacterianos de mastites bovinas; avaliar a capacidade dos fagos isolados de inibir e remover biofilmes bacterianos de isolados oriundos de mastites bovinas e cepas bacterianas. Na seção correspondente ao Capítulo 1 desta tese, apresenta-se um artigo científico de meta-análise, realizado com objetivo de investigar a eficiência de vários fagos de S. aureus frente a diferentes bactérias oriundas de mastites bovinas, confirmando a alta eficiência (80%) e especificidade da maioria dos fagos dos estudos incluídos na meta-análise. A seguir, no Capítulo 2, no manuscrito 1 objetivou-se realizar o isolamento de fagos oriundos de leite bovino. Estes fagos foram denominados B_UFSM1, B_UFSM3, B_UFSM4, e B_UFSM5 e, a partir da caracterização genômica e fenotípica de dois fagos temperados de S. aureus (B_UFSM4 e B_UFSM5), verificou-se que os mesmos pertencem à ordem Caudovirales, família Siphoviridae e gênero Biseptimavirus. Adicionalmente, com análise da eficiência de plaqueamento, verificou-se que os fagos B_UFSM4 e B_UFSM5 são capazes de infectar isolados de sua espécie hospedeira alvo e, também, possuem a habilidade de infectar outra espécie bacteriana, Rothiae terrae. Em seguida, no Capítulo 3, referente ao manuscrito 2, foi investigado se os fagos e duas combinações A (B_UFSM4 e B_UFSM5) e B (B_UFSM1, B_UFSM3, B_UFSM4 e B_UFSM5), apresentavam a capacidade de inibir e remover biofilmes bacterianos de Staphylococcus spp. e de Pseudomonas aureginosa oriundos de mastite bovina, bem como, de cepas-padrão bacterianas. Neste sentido, foi possível observar que os fagos e as combinações tiveram atividade antibiofilme e somente o fago B_UFSM4 não foi capaz de inibir a formação e/ou remover os biofilmes dos isolados e cepas-padrão bacterianas testadas. Dessa forma, conclui-se, que os fagos isolados, caracterizados e analisados deverão ser mais estudados para verificar o potencial uso como alternativas terapêutica clínica e no controle de biofilmes de S. aureus oriundos de mastite bovina. Adicionalmente, devido a pandemia COVID-19, ocorreram atrasos nos prazos estabelecidos para a finalização da avaliação e caracterização morfológica e molecular, dos fagos B_UFSM1 e B_UFSM3; sendo assim, estes resultados não puderam ser incluídos nesta tese.
Bacteriophages or phages are viruses that have the ability to infect and lyse bacteria through cell lysis. These viruses may represent a promising alternative for the treatment of bacterial infections, especially those caused by strains multiresistant to antimicrobial therapies, such as bovine mastitis by Staphylococcus aureus and in control of bacterial biofilms. Therefore, the objectives of this study were: isolating and characterizing S. aureus phages from bovine milk; test the efficiency of phages against bovine mastitis bacterial isolates; evaluating the ability of isolated phages to inhibit and remove bacterial biofilms from bovine mastitis isolates and standard bacterial strains. In the section corresponding to Chapter 1 of this thesis, a scientific article of meta-analysis is presented, carried out with the aim of investigating the efficiency of several S. aureus phages against different bacteria from bovine mastitis, confirming the high efficiency (80%) and specificity of most phages from the studies included in the meta-analysis. Subsequently, in Chapter 2, in manuscript 1, aimed to isolate phages from bovine milk. These phages were named B_UFSM1, B_UFSM3, B_UFSM4, and B_UFSM5 and, with the genomic and phenotypic characterization of two temperate S. aureus phages (B_UFSM4 and B_UFSM5) it was found that they belong to the order Caudovirales, family Siphoviridae and Biseptimavirus genus. In addition, with the analysis of plating efficiency, it was found that the phages B_UFSM4 and B_UFSM5 are able to infect their target host species e also have the ability to infect another bacterial species, as Rothiae terrae. Then, in Chapter 3, referring to manuscript 2, it was investigated whether phages and two combinations A (B_UFSM4 and B_UFSM5) and B (B_UFSM1, B_UFSM3, B_UFSM4 and B_UFSM5) had the ability to inhibit and remove bacterial biofilms of Staphylococcus spp. and Pseudomonas aeruginosa from bovine mastitis, as well as standard bacterial strains. It was possible to observe that the phages and combinations had antibiofilm activity and only the B_UFSM4 phage was not able to inhibit the formation and/or remove the biofilms of the tested bacterial isolates and standard strains. Thus, it is concluded that the isolated, characterized and analyzed phages should be further studied to verify their potential use as clinical therapeutic alternatives and in the control of S. aureus biofilms from bovine mastitis. Additionally, due to the COVID-19 pandemic, there were delays in the deadlines established for the completion of the morphological and molecular evaluation and characterization of the phages B_UFSM1 and B_UFSM3; therefore, these results could not be included in this thesis.
Resumo
A infecção por bactérias como Salmonella enterica e Escherichia coli, é uma ameaça à saúde humana, sendo as principais causadoras de doenças transmitidas por alimentos (DTAs) e de resistência à antibióticos. Neste contexto, a utilização de bacteriófagos vem tornando uma alternativa promissora que oferece muitas vantagens, como à especificidade do vírus, encontrado naturalmente em diversos ambientes, ao alto grau de segurança e à potência. O objetivo deste trabalho foi isolar e caracterizar fagos líticos que efetivamente controlam a contaminação de alimento por bactérias de importância alimentar. Para tanto, nove fagos foram isolados a partir de amostras de águas residuárias oriundas de ambiente industrial, utilizando cepas padrão de Salmonella enterica sorovar Enteritidis (S. Enteritidis), Salmonella enterica sorovar Typhimurium (S. Typhimurium) e Escherichia coli, através do método de isolamento e purificação. Dentre estes, três bacteriófagos (ST.22, SE.21 e EC.01.03) foram eleitos por demonstrarem uma maior atividade lítica contra as bactérias hospedeiras avaliadas (S. typhimurium, S. enteritidis e E. coli, respectivamente). Os bacteriófagos foram capazes de controlar os respectivos hospedeiros na avaliação in loco de contaminação intencional em linguiça frescal, reduzindo a contagem final de bactérias viáveis em aproximadamente 1,0 log10 UFC/mL a 4°C em relação ao controle bacteriano. Individualmente, os fagos ST.22 e EC 01.03 demonstraram maior eficiência lítica sobre as bactérias remanescentes quando comparadas ao controle negativo. Em destaque, a incubação do fago ST.22 a 4°C reduziu entre 1,0 log10 UFC/mL e 3,0 log10 UFC/mL na contagem final de bactérias viáveis (S. Enteritidis/E.coli e S. Typhimurium, respectivamente). A ação do fago EC 01.03 na linguiça frescal a 4°C promoveu a redução de todas as bactérias em 1,0 log10 UFC/mL em relação ao controle negativo. Contudo, são necessários estudos adicionais para melhor conhecimento da gama de hospedeiros de tais bacteriófagos, ampliando sua caracterização, etapa essencial para o desenvolvimento de aplicação efetiva contra Escherichia coli e Salmonella patogênica em alimentos prontos para consumo.
Infections by bacteria such as Salmonella enterica and Escherichia coli, are one of the greatest threats to human health, being the main cause of foodborne diseases and resistance to antibiotics. They are responsible for causing great economic losses in the global scenario, becoming a constant challenge for the food industry. In this context, the use of bacteriophages has become a promising alternative, which offers many advantages over conventional chemical preservatives, such specificity of the pathogen, found naturally in various environments, with a high degree of safety and power. The objective of this work was to isolate and characterize lytic phages that effectively control the contamination of food by bacteria of food importance. For this purpose, 9 phages were isolated, obtained from wastewater samples from industrial environments, using standard strains of Salmonella enterica sorovar Enteritidis (S. enteritidis), Salmonella enterica sorovar Typhimurium (S. typhimurium) and Escherichia coli, through the isolation and purification method. Among these, 3 bacteriophages (ST.22, SE.21 and EC.01.03) were determined by demonstrating a higher lytic activity against the evaluated host and non-host strains, coming from S. typhimurium, S. enteritidis and E. coli, respectively. Therefore, the capacity of both defined phages were able to control the hosts themselves in frescal sausage in the on-site evaluation of intentional food contamination, reducing the final viable bacteria count by approximately 1.0 log10 CFU/mL at 4ºC in relation to the phage excluded control. At individual performance, ST.22 and EC 01.03 demonstrated greater lytic efficiency in controlling the remaining bacteria in the test when compared to the negative control in the final disposition. In prominence, the incubation of ST.22 phage at 4ºC reduced between 1.0 log10 CFU/mL and 3.0 log10 CFU/mL in the final count of viable bacteria non-host to host, respectively. And with the administration of EC 01.03 in frescal sausage, the count of viable bacteria, host independent or not, decreased exactly 1.0 log10 CFU/mL at 4ºC, in relation to the negative control. Nonetheless, further studies are needed to evaluate the range of hosts of such bacteriophages, broadening their characterization, an essential step for the development of effective application against Escherichia coli and pathogenic Salmonella against various ready-to-eat foods.
Resumo
Doenças de veiculação hídrica são um problema de saúde pública, pois a presença de bactérias coliformes na água, particularmente os coliformes termotolerantes como Escherichia coli, é um dos indicadores de contaminação fecal. Logo, o uso de bactérias como o único indicador microbiológico de qualidade hídrica vem sendo revisto, principalmente pela existência de diferentes patógenos, como vírus e protozoários. Embora a quantificação de colifagos, vírus que infectam E. coli, como parâmetro de qualidade microbiológica da água seja reconhecida pela American Public Health Association (APHA), esta não é uma prática em nosso país. Por isso, este estudo avaliou a utilidade de colifagos como indicadores de contaminação fecal em amostras de água bruta e tratada e em amostras de esgoto bruto e tratado em estações, comparando-os com os resultados dos indicadores clássicos (coliformes). As seis coletas ocorreram entre maio e agosto de 2019 e as amostragens foram realizadas junto às estações de tratamento de Novo Hamburgo (RS). Todas amostras foram previamente submetidas à etapa de concentração viral para a pesquisa de fagos, exceto as amostras de água pós-tratamento, pois estas foram enriquecidas. Foi realizado o ensaio em sobrecamada de ágar para observação de placas de lise mediante três experimentos independentes para cada amostra de água e esgoto, todos em duplicata, utilizando Escherichia coli ATCC 13706 como célula hospedeira. Todos os tipos de amostras (água e esgoto, bruto e tratado) mostraram-se positivos para a presença de fagos, mas não para coliformes. Enquanto todas as amostras de água pós-tratamento foram negativas para coliformes, observou-se a presença de placas de lise nos ensaios realizados em 33,3% (2/6) dessas amostras, evidenciando que os colifagos são indicadores microbiológicos diferenciados. Na água tratada, a etapa de enriquecimento da fonte de isolamento foi muito importante para observação das placas de lise no teste em sobrecamada de ágar. Assim, os resultados demonstraram que os colifagos são resistentes ao tratamento físico-químico utilizado na respectiva estação, reforçando a sua utilidade como indicadores da contaminação por material fecal, especialmente em amostras de água tratada.
Waterborne diseases are a concern in public health system, since the presence of coliform bacteria in the water, particularly the thermotolerant coliforms/ fecal coliforms Escherichia coli is one of the indicators of fecal contamination. Therefore, the use of bacterias as the only microbiological indicator of water quality is being revised, mainly due to the existence of different pathogens, such as viruses and protozoans. Although the coliphage quantification, the virus which infects E. coli, as a microbiological quality standard of water is recognized by the American Public Health Association (APHA), this is not a practice in our country. Therefore, this study evaluated the coliphage usefulness as indicators of fecal contamination in raw and treated water samples, as well as in samples of raw sewage and treated plants, comparing them with the results of the classical indicators (coliforms). The six collections occurred between May and August of 2019 and the sampling processes were held together with the treatment plants of Novo Hamburgo (RS). All the samples were previously submitted to a viral concentration method to search for phages, besides the post-treatment water samples, for these have been enriched. It was held an agar-layer method to observe the lysis plates through three independent experiments for each water and sewage sample, all in duplicata, using Escherichia coli ATCC 13706 as the host cell. All sample types (water, sewage, raw, and treated) turned out to be positive for the presence of phages, but not to coliforms. While all the post-treatment water samples were negative for coliforms, it was observed the presence of lysis plates in tests performed in 33,3% (2/6) of these samples, emphasizing that the coliphages are differentiated microbiological indicators. In treated water, the enrichment of the insulation source method was very important to the observation of lysis plates in the agar-layer method. Thus, the results showed that the coliphages are resistant to the physicochemical treatment in the respective plant, reinforcing their usefulness as indicators of contamination through fecal material, especially in treated water samples.
Resumo
A fagoterapia é um método de controle de infecções conhecido há mais de cem anos, porém deixou-se de ser utilizada devido à praticidade do uso de antibióticos. No entanto, com o aumento mundial da resistência bacteriana, a fagoterapia ressurge como opção terapêutica, porém os padrões de seleção de amostras utilizados são empíricos. O presente artigo descreve o comportamento in vitro de 62 amostras de bacteriófagos líticos isolados de esgoto hospitalar humano usando uma amostra de Salmonella Enteritidis de origem avícola. No estudo, foram testados quatro perfis distintos comumente utilizados nas pesquisas com bacteriófagos e observou-se que há uma variância comportamental considerável em que atividade lítica, tamanho de halo em gel difusão e índice de proliferação são fatores independentes de virulência, porém amostras prolíferas apresentam maior espectro de atividade lítica.
This article analyzed, in vitro, the behavior of 62 bacteriophages samples lytic for Salmonella Enteritidis. The lytic potential was assessed by analyzing following parameters: proliferation, halo size in gel diffusion, superficial halo size and virulence degree. Proliferation degree of samples was determined by quantifying the amount of plaque forming unit (PFU) using gel diffusion technique. To determine UFP, minor dilution with measurable halos was considered. Lytic spectrum in the gel column was measured with an acrylic precision ruler. For to superficial halo size and virulence degree, the "spot-on-lawn" technique was employed. We observed that lytic activity spectrum, halo size in gel diffusion and proliferation are factors independent of virulence, but prolific samples shown higher spectrum of lytic activity. Prolific samples that present a high degree of virulence demonstrate in vitro a good potential for medical application.
La fagoterapia es un método de control de la infección conocido por más de cien años, sin embargo, su uso se dejó de aplicar debido a la practicidad de los antibióticos. Con el aumento mundial de la resistencia bacteriana, la fagoterapia resurge como una opción terapéutica, pero los patrones de selección de muestras utilizadas son empíricas. En este trabajo, se describe el comportamiento in vitro de 62 muestras de los bacteriófagos líticos aislados de aguas residuales del hospital humano utilizando una muestra de Salmonella Enteritidis original de productos avícolas. En el estúdio, se probaron cuatro diferentes perfiles comúnmente utilizados en la investigación en bacteriófagos y se observó que existe una variación considerable en donde el espectro de comportamiento de actividad lítica, tamaño de halo en gel difusión y el índice de proliferación fueron factores independientes de la virulencia, pero las muestras prolíficos tienen un mayor espectro de acción.
Assuntos
Salmonella/virologia , Fagos de Salmonella/isolamento & purificação , Bacteriófagos/classificação , Terapia por Fagos/tendênciasResumo
A constante busca pelo aperfeiçoamento nos processos que garantam a segurança dos alimentos impulsionam estudos com o intuito de conhecer melhor as características de patógenos, que, quando presentes, causam impactos negativos ao setor avícola. Salmonella Heidelberg (SH) é o sorovar que se destacou nos últimos anos devido sua alta prevalência e multirresistência aos antimicrobianos. Buscou-se nesse trabalho informações quanto à formação de biofilmes, à suscetibilidade antimicrobiana, e à detecção de genes de virulência, permitindo, assim, melhor entendimento e maneiras alternativas de eliminação da SH. Encontramos variados perfis genotípicos (56), diferentes graus de formação de biofilme e altas taxas de multirresistência (125/126 - 99,2%), sugerindo que há uma diversidade de isolados de SH em produtos avícolas, o que pode indicar a existência de fontes de contaminação variáveis na indústria de alimentos. Atualmente, o mundo está enfrentando um crescente surgimento de bactérias resistentes aos antibióticos e a necessidade de reduzir o seu uso indiscriminado é fundamental. A procura de novos compostos antimicrobianos a partir de espécies vegetais, como óleos essenciais (OEs), e o uso de bacteriófagos (fagos), tem se mostrado bastante expressiva. Os fagos são predadores naturais de bactérias, onipresentes no ambiente e com alta especificidade ao hospedeiro, inofensivo para homens e animais. Os OEs são utilizados desde a idade média nos mais diferentes ramos das indústrias farmacêutica, sanitária, agrícola e de alimentos, e sua atividade bactericida também é tida como destaque. Nesse contexto, ambos são considerados alternativas valiosas e uma oportunidade de reduzir o uso de antimicrobianos. Avaliou-se a capacidade de biocontrole com o uso de três bacteriófagos (UPF_BP1, UPF_BP2, UPF_BP3) isolados ou combinados (pool) para prevenção e remoção de biofilmes de SH em superfície de poliestireno, em tempos de 3, 6 e 9 horas de ação. Os fagos individuais e em combinação demonstraram reduções na adesão de SH em até 83,4%, e remoção do biofilme pré-formado de até 64,0%. Ressalta-se que o uso de combinações sinérgicas entre os fagos é a mais indicada por potencializar a redução, bem como o uso no condicionamento de superfícies ser mais eficaz do que em biofilmes pré-formados. No teste com os OEs, avaliou-se a ação in vitro de cinco óleos (orégano, canela, tomilho, cravo e gengibre) sobre os isolados de SH, avaliando atividade antimicrobiana em ágar difusão e concentração inibitória mínima (CIM). O orégano e a canela obtiveram ação antimicrobiana em baixas concentrações sobre os isolados. Além disso, o efeito do OE de orégano foi testado em matriz cárnea nas temperaturas de 12±1ºC e a 4±1ºC por 0, 1, 3 e 5 dias, sendo eficiente para redução da carga microbiana em diferentes temperaturas e tempo de contato com a matriz confrontada, enfatizando a importância da associação à baixas temperaturas. Com esses resultados temos disponíveis métodos naturais que podem ser alternativas para o controle desse patógeno, e contribuem para o futuro uso de bacteriófagos como medida de condicionamento de superfícies no biocontrole de biofilmes de SH.
The constant search for improvement in processes that guarantee food safety, stimulate studies in order to better understand pathogens characteristics, if present, causes negative impacts to poultry sector. Salmonella Heidelberg (SH) is the sorovar that has stood out in recent years due to its high prevalence and antimicrobials resistence. The objective of this research was to find information regarding the biofilm formation, antimicrobial susceptibility, and virulence genes detection, thus allowing a better understanding and alternative ways to eliminate SH. We found varied genotypic profiles (56), different biofilm formation degrees and high multi-resistance rates (125/126 -99.2%), suggesting that there is a diversity of SH isolates in poultry products, which may indicate existence of sources contamination at food industry. Currently, the world is facing an increasing emergence of antibiotic resistant bacteria and the need to reduce their indiscriminate use is fundamental. The search for new antimicrobial compounds from plant species, such as: the essential oils (EOs) and also the use of bacteriophages (phages), has been shown to be expressive. Phages are natural predators of bacteria, they are ubiquitous in environment and with high specificity to the host, harmless to man and animals. EOs have been used since middle ages in most different branches of pharmaceutical, sanitary, agricultural and food industries, and their bactericidal activity is also highlighted. In this context, both are considered valuable antimicrobial alternatives and an opportunity to reduce the use of antimicrobials. The biocontrol ability was evaluated using three bacteriophages (UPF_BP1, UPF_BP2, UPF_BP3) isolated or combined (pool) to prevent and remove SH biofilms on polystyrene surfaces, in 3, 6 and 9 h of action. Individual and combined phages showed reductions in SH adherence up to 83.4%, and preformed biofilm removal up to 64.0%. It´s noteworthy that the use of synergistic combinations between phages is most indicated, since it has enhanced reduction, as well as use in surface conditioning to be more effective than in preformed biofilms. In test with EOs, in vitro action of five oils (oregano, cinnamon, thyme, cloves and ginger) on SH isolates was evaluated, evaluating antimicrobial activity in diffusion agar, minimum inhibitory concentration (MIC). Oregano and cinnamon obtained antimicrobial action in low concentrations on isolates. In addition, the effect of EO of oregano was tested in a meat matrix at temperatures of 12±1ºC and at 4±1ºC for 0, 1, 3 and 5 days, being efficient for reducing the microbial load at different temperatures and time of contact with the confronted matrix, emphasizing the importance of association with low temperatures. With these results we have available natural methods that can be alternatives for pathogen control and contribute to future use of bacteriophages as a measure of surface conditioning in SH biofilms biocontrol. With these results we have available natural methods that can be alternatives for pathogen control and contribute to future use of bacteriophages as a measure of surface conditioning in SH biofilms biocontrol.
Resumo
O tifo aviário possui grande importância econômica em função dos altos custos com programas de controle, devido ao sacrifício sanitário das aves e descarte dos ovos nos incubatórios. A Salmonella Gallinarum, há algumas décadas, havia sido considerada erradicada em aves comerciais no Brasil. Porém, acredita-se que, devido às falhas nos controles, como biosseguridade, insumos, monitorias e programas sanitários, associados aos mecanismos de virulência do patógeno, essa doença vem reemergindo nos últimos anos. A S. Gallinarum causa infecção e manifestações clinicas, com alta concentração bacteriana nos tecidos e pode ser transmitida vertical e horizontalmente. A contaminação via transovariana torna os ovos inviáveis para a produção de aves, as quais podem se tornar portadoras assintomáticas e transmitir o patógeno logo após o nascimento. A transmissão do microrganismo por via horizontal pode se caracterizar por contaminar os ovos através da casca logo após a postura, nos ninhos ou camas de aviários. Os fatores de virulência influenciam nos tratamentos com drogas antimicrobianas e no controle ambiental com a utilização de desinfetantes. É de fundamental importância o estudo desses fatores para auxiliar na análise epidemiológica dos surtos ocorridos e, também, para estabelecer estratégias de novos controles. O objetivo deste estudo foi realizar a caracterização fenotípica e genotípica de diferentes isolados de S. Gallinarum, oriundos da produção agroindustrial, através da avaliação da sensibilidade aos antimicrobianos e a desinfetante, potencial de lise por bacteriófagos, pesquisa de genes de virulência, formação de biofilme em poliestireno e em cascas de ovos. Pode-se observar que as S. Gallinarum são multirresistentes aos antimicrobianos, sensíveis aos bacteriófagos testados, e potencialmente virulentas, de acordo com perfis genéticos obtidos. A maioria dos isolados formaram biofilme no poliestireno tanto a 22°C como a 42°C, sem diferença estatística (p = 0.0965) entre as temperaturas testadas. Houve maior formação de biofilme nas cascas de ovos na temperatura de 22°C (4,656 log10UFC/cm2), com diferença estatística quando comparado aos biofilmes formados a 36°C e 42°C. A ação do desinfetante comercial, na concentração 1,5% e tempo 5 minutos, promoveu remoção do biofilme, estatisticamente significativa, apenas na formação a 22°C, propiciando uma redução de 3,125 log10. A presença de genes de virulência, a multirresistência e a capacidade de formar biofilmes em diferentes superfícies e temperaturas pelas S. Gallinarum estudadas, isoladas de aves comerciais, nos faz supor que estes podem ser fatores relevantes para que a SG se mantenha presente nos plantéis avícolas e esteja envolvida em surtos sanitários intermitentes. Apesar do cenário preocupante, a possibilidade de controle biológico da SG por bacteriófagos nos traz uma alternativa promissora para a contenção deste microrganismo.
The fowl typhoid has great economic importance due to the high costs with control programs, due to the sanitary sacrifice of the birds and discard of the eggs in the hatcheries. Salmonella Gallinarum, a few decades ago, had been considered to be eradicated in commercial birds in Brazil. However, due to the lack of controls, such as biosecurity, inputs, monitoring and health programs, associated with the virulence mechanisms of the pathogen, this disease has been reemerging in recent years. S. Gallinarum causes infection and clinical manifestations, with high bacterial concentration in the tissues and can be transmitted vertically and horizontally. The contamination via transovarian renders the eggs unviable for the production of birds, which can become asymptomatic carriers and transmit the pathogen soon after the birth. Transmission of the microorganism horizontally can be characterized by contaminating the eggs through the bark soon after laying, in the nests or beds of aviaries. Virulence factors influence the treatments with antimicrobial drugs and environmental control with the use of disinfectants. It is of fundamental importance the study of these factors to assist in the epidemiological analysis of the outbreaks occurred and, also, to establish strategies of new controls. The objective of this study was to characterize the phenotypic and genotypic characteristics of different S. Gallinarum isolates from agroindustry production through the evaluation of antimicrobial susceptibility and disinfectant, bacteriophage lysis potential, virulence genes, biofilm formation Polystyrene and eggshells. It can be observed that the S. Gallinarum are multiresistant to the antimicrobials, sensitive to the bacteriophages tested, and potentially virulent, according to the genetic profiles obtained. Most of the isolates formed biofilm in polystyrene at 22°C and 42°C, with no statistical difference (p = 0.0965) between the temperatures tested. There was a higher biofilm formation in eggshells at 22°C (4,656log10UFC / cm2), with a statistical difference when compared to biofilms formed at 36°C and 42°C. The action of the commercial disinfectant, in the concentration 1.5% and time 5 minutes, promoted the removal of the biofilm, statistically significant, only in the formation at 22°C, leading to a reduction of 3,125 log10. The presence of virulence genes, the multiresistance and the ability to form biofilms on different surfaces and temperatures by S. Gallinarum studied, isolated from commercial birds, makes us suppose that these can be relevant factors for the SG to remain present in poultry farms and is involved in intermittent outbreaks. Despite the worrying scenario, the possibility of biological control of SG by bacteriophages brings us a promising alternative for the containment of this microorganism.
Resumo
Salmonella enterica serovar Enteritidis (SE) is a prevalent gastrointestinal pathogen worldwide, threatening both animal and human health. In the latter, disease is associated to the consumption of SE-contaminated products from the poultry industry. The control of SE infection is largely based on the use of antibiotics and vaccines, but the use of lytic bacteriophages is re-emerging as an additional strategy for SE control. In fact, a number of recent reports point to the adequacy of bacteriophage as an efficient prophylactic or therapeutic countermeasure to SE infections. However, less attention has been focused on the basic biology of these bacteriophages. Here we report on three bacteriophages (f18, IF1 and EST2) that share a common viral particle morphology but are genomic variants as judged by their EcoRI DNA restriction patterns. Furthermore, they differ in their lytic capability towards SE, being EST2 the most efficient. They show a very narrow host range, efficiently infecting only SE strains. In terms of stability in various suspension media, including distilled water, all three bacteriophages remained viable, without noticeable decay in titer for at least 15 days at 25ºC. These results suggest the suitability of the tested bacteriophages as SE-controlling agents in the poultry industry.
Resumo
Salmonella enterica serovar Enteritidis (SE) is a prevalent gastrointestinal pathogen worldwide, threatening both animal and human health. In the latter, disease is associated to the consumption of SE-contaminated products from the poultry industry. The control of SE infection is largely based on the use of antibiotics and vaccines, but the use of lytic bacteriophages is re-emerging as an additional strategy for SE control. In fact, a number of recent reports point to the adequacy of bacteriophage as an efficient prophylactic or therapeutic countermeasure to SE infections. However, less attention has been focused on the basic biology of these bacteriophages. Here we report on three bacteriophages (f18, IF1 and EST2) that share a common viral particle morphology but are genomic variants as judged by their EcoRI DNA restriction patterns. Furthermore, they differ in their lytic capability towards SE, being EST2 the most efficient. They show a very narrow host range, efficiently infecting only SE strains. In terms of stability in various suspension media, including distilled water, all three bacteriophages remained viable, without noticeable decay in titer for at least 15 days at 25ºC. These results suggest the suitability of the tested bacteriophages as SE-controlling agents in the poultry industry.
Resumo
Reducing Salmonella contamination in poultry is of major importance to prevent the introduction of this microorganism into the food chain. Salmonellae may spread during storage time (shelf life) whenever pre-harvest control fails or post-harvest contamination occurs. Therefore, preventive measures should also be used in the post-harvest level of poultry production in order to control salmonellae. Chicken skin samples were experimentally contaminated by immersing whole legs (thighs and drumsticks) in a suspension containing 10(6) colony forming units per milliliter (CFU/mL) of Salmonella Enteritidis phage type 4 (SE PT4) at the slaughter day. One day later, samples from one group were immersed in a suspension pool containing 10(9) CFU/mL of each of three wild salmonella-lytic bacteriophages previously isolated from feces of free-range chickens. Salmonella counting was performed at three-day intervals in the chicken legs stored at 5°C and showed a significant reduction (P 0.05) of SE PT4 in bacteriophage-treated cuts on days 3, 6 and 9 post-treatment. These findings suggest that the use of bacteriophages may reduce SE PT4 in chicken skin. Further studies are encouraged and might demonstrate the potential of this approach as an efficient and safe technique to be routinelly used for Salmonella control in chicken products.
Resumo
Reducing Salmonella contamination in poultry is of major importance to prevent the introduction of this microorganism into the food chain. Salmonellae may spread during storage time (shelf life) whenever pre-harvest control fails or post-harvest contamination occurs. Therefore, preventive measures should also be used in the post-harvest level of poultry production in order to control salmonellae. Chicken skin samples were experimentally contaminated by immersing whole legs (thighs and drumsticks) in a suspension containing 10(6) colony forming units per milliliter (CFU/mL) of Salmonella Enteritidis phage type 4 (SE PT4) at the slaughter day. One day later, samples from one group were immersed in a suspension pool containing 10(9) CFU/mL of each of three wild salmonella-lytic bacteriophages previously isolated from feces of free-range chickens. Salmonella counting was performed at three-day intervals in the chicken legs stored at 5°C and showed a significant reduction (P 0.05) of SE PT4 in bacteriophage-treated cuts on days 3, 6 and 9 post-treatment. These findings suggest that the use of bacteriophages may reduce SE PT4 in chicken skin. Further studies are encouraged and might demonstrate the potential of this approach as an efficient and safe technique to be routinelly used for Salmonella control in chicken products.
Resumo
Occurrence of food poisoning related to Salmonella-contaminated eggs and chicken meat has been frequent in humans. Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) are included among the most important paratyphoid salmonellae associated with chicken meat and eggs. Elimination of Salmonella at the pre-harvest stage can play a significant role in preventing the introduction of this pathogen into the food chain and consequently in the reduction of food poisoning in humans. Bactericidal bacteriophages may provide a natural, nontoxic, feasible and non-expensive component of the multi-factorial approach for a pre-harvest control of Salmonella in poultry. Five bacteriophages lytic for SE PT4 and ST were obtained from 107 samples of feces of free-range layers in Brazil. All bacteriophages were characterized in vitro and in vivo, showing head and tail morphology and dsDNA as nucleic acids. Results of "in vivo" studies suggested that bacteriophages do not remain in Salmonella-free birds longer than one day, whereas they multiply in Salmonella-infected birds for longer periods. Besides, selection for phage-resistant SE PT4 did not seem to occur in the short term. Isolated bacteriophages will be investigated for their potential for pre-harvest biocontrol of SE PT4 in poultry.
Resumo
Occurrence of food poisoning related to Salmonella-contaminated eggs and chicken meat has been frequent in humans. Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) are included among the most important paratyphoid salmonellae associated with chicken meat and eggs. Elimination of Salmonella at the pre-harvest stage can play a significant role in preventing the introduction of this pathogen into the food chain and consequently in the reduction of food poisoning in humans. Bactericidal bacteriophages may provide a natural, nontoxic, feasible and non-expensive component of the multi-factorial approach for a pre-harvest control of Salmonella in poultry. Five bacteriophages lytic for SE PT4 and ST were obtained from 107 samples of feces of free-range layers in Brazil. All bacteriophages were characterized in vitro and in vivo, showing head and tail morphology and dsDNA as nucleic acids. Results of "in vivo" studies suggested that bacteriophages do not remain in Salmonella-free birds longer than one day, whereas they multiply in Salmonella-infected birds for longer periods. Besides, selection for phage-resistant SE PT4 did not seem to occur in the short term. Isolated bacteriophages will be investigated for their potential for pre-harvest biocontrol of SE PT4 in poultry.
Resumo
Over the last few years, a number of strategies to minimize the microbial load in raw products have beenexplored. Problems with the acceptability and deterioration of organoleptic properties have been describedafter physical treatments such as steam, dry heat and UV light. Moreover, consumers are looking for morenatural foods, i.e. free from chemical preservatives. An approach that has been the object of growing interestis the use of bacteriophages, which are viruses that infect and kill bacteria. Bacteriophages are components ofthe natural microflora in the food production continuum from the farm to the retail outlet; they are stable inthese environments and are readily recovered from soil, sewage, water, farm and processing plant effluents,feces, and retail foods. Studies on pathogens control in foods by using bacteriophage are promising, howeverthey have to be improved. The success of this methodology will depend on the consumers acceptance. Inthis way, it is recommended to carefully consider the origin of the phage before introducing it into foods.
Nos últimos anos, um grande número de estratégias para minimizar a contaminação microbiana dealimentos tem sido explorado. Problemas com aceitabilidade e alteração nas características organolépticasdos alimentos, decorrentes de tratamentos físicos como o emprego do calor e da luz ultravioleta, têm sidodescritos. Além disso, os consumidores estão buscando cada vez mais os alimentos naturais, isto é, isentosde conservantes químicos. Uma abordagem que tem sido objeto de interesse crescente é a utilização debacteriófagos, que são vírus que infectam e matam as bactérias. Os bacteriófagos são componentes damicrobiota natural da produção de alimentos, desde o campo até a comercialização; são estáveis nessesambientes e são prontamente recuperados do solo, da água, de efluentes, das fezes e dos próprios alimentos.Os estudos para o controle de micro-organismos patogênicos em alimentos com o uso de bacteriófagos sãopromissores, mas ainda necessitam ser aperfeiçoados. O sucesso dessa metodologia dependerá da aceitaçãodo consumidor dentre outros fatores. Por esta razão, é prudente considerar cuidadosamente a fonte dequalquer bacteriófago antes deste ser aplicado nos alimentos.