Enhanced killing of multidrug-resistant Pseudomonas aeruginosa ATCC10145 through a combined action of antibiotics and bacteriocin from Pediococcus pentosaceus TU2

Aims@#Due to its rapid development of resistance against most conventional antibiotics, there is an urgent need to develop new antimicrobial agents and strategies to overcome the challenges in combating multidrug-resistant Pseudomonas aeruginosa infections. This study aimed to determine the antipseudomonal potency of bacteriocin produced by Pediococcus pentosaceus TU2 when combined with conventional antibiotics.@*Methodology and results@#The checkerboard method and time-kill assay were conducted to investigate the antagonism interaction and kinetics of the bacteriocin TU2 and selected antibiotics against Pseudomonas aeruginosa ATCC10145. The scanning electron microscope (SEM) was used to observe the cell surface morphological changes of the treated P. aeruginosa ATCC10145. The combination of bacteriocin TU2 with ciprofloxacin and tetracycline resulted in a 4-fold reduction in minimum inhibitory concentration (MIC) and a fractional inhibitory concentration index (ΣFICI) of 0.5, indicating a synergistic interaction against P. aeruginosa ATCC10145. Similarly, the time-kill assay showed that the combination of bacteriocins TU2 respectively with chloramphenicol and tetracycline exerted enhanced bactericidal effect at 8 h and 10 h of treatments compared to treatment with antimicrobial agents alone. Results from SEM suggested that bacteriocin TU2 might cause pore formation on cells and thus enhanced the membrane permeability of antibiotics and intensified the membrane leakage that led to cell death of P. aeruginosa ATCC10145.@*Conclusion, significance and impact of study@#The combined antagonistic effect of bacteriocin TU2 and antibiotics could be a promising strategy in combating P. aeruginosa infections and may be applied in therapeutic industries.

Pediococcus pentosaceus as probiotic with cholesterol-lowering ability

Aims@#Hypercholesterolemia which is an elevated blood cholesterol level that considered as a major risk factor for cardiovascular disease, which is the leading cause of death in many countries. Therefore, lowering the cholesterol level is important to prevent the disease. Lactic acid bacteria (LAB) group are often used as probiotics for their healthpromotion which include cholesterol-lowering effect. The purpose of this study was to evaluate the potency of Pediococcus pentosaceus as probiotic that could reduce cholesterol. @*Methodology and results@#All P. pentosaceus strains were able to survive in acid conditions and in the presence of 0.3% bile salts. These strains had antimicrobial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Salmonella typhimurium ATCC 14028. The LAB were also sensitive to chloramphenicol and showed autoaggregation and coaggregation ability. Pediococcus pentosaceus E5, E7, and E8 were able to remove cholesterol with the highest activity showed by P. pentosaceus E7 (49.00 ± 2.83%). Dead cells and resting cells of P. pentosaceus E5, E7, and E8 (6-22%) also able to reduce the cholesterol but not as effectively as growing cells. Cholesterol lowering is often associated with bile salt hydrolase (BSH) enzyme activity, however none of the isolates were found BSH positive in this study. @*Conclusion, significance and impact of study@#The present study suggests that P. pentosaceus E7 has beneficial probiotic properties which can be exploited for probiotic product with cholesterol-lowering effect.

Bioprodução de pediocina por Pediococcus pentosaceus ET34 em resíduo agroindustrial hidrolisado bagaço de cana-de-açúcar / Bioproduction of Pediocin by Pediococcus pentosaceus ET34 in hydrolyzed sugar cane bagasse agroindustrial residue

Um dos grandes desafios nas indústrias alimentícia, farmacêutica e agropecuária é a busca por novos compostos para substituir os antibióticos. Como possíveis candidatos estão as bacteriocinas para serem utilizados paralelamente aos antibióticos ou até substituí-los. As bactérias ácido-láticas podem produzir substâncias inibitórias semelhantes às bacteriocinas (BLIS - bacteriocin-like inhibitory substances) que possuem efeito bacteriostático ou bactericida contra diferentes grupos de bactéria sendo largamente utilizadas como bioconservantes alimentares. Neste contexto, o objetivo deste trabalho foi o emprego de um resíduo agroindustrial, o hidrolisado de bagaço de cana-de-açúcar, como fonte de carbono em cultivos fermentativos para produção do BLIS pela cepa Pediococcus pentosaceus ET 34. Resultados revelaram que as células de ET34 foram capazes de crescer utilizando este resíduo agroindustrial como fonte de carbono e ensaios utilizando planejamento fatorial demonstraram que a agitação não influencia na produção de BLIS. Ao escalonar o cultivo em biorreatores, foi verificado que tanto o crescimento como a atividade antimicrobiana foram semelhantes aos obtidos em bancada com exceção da condição de 25% (v/v) de HBC (hidrolisado de bagaço de cana) que devido a maior viscosidade do meio, resultou em uma diminuição no crescimento e de produção de BLIS. O BLIS produzido por células de ET34 utilizando o HBC como fonte de carbono foi parcialmente purificado por sulfato de amônio e demonstrou atividade contra Listeria monocytogenes ATCC 934 e Salmonella enterica CECT 724. Desta maneira, pode-se concluir que Pediococcus pentosaceus ET34 foi capaz de crescer em meio contendo HBC como fonte de carbono produzindo BLIS em seu sobrenadante com ação frente a diferentes bactérias patogênicas. A possibilidade de utilizar uma fonte alternativa de carbono pode diminuir o custo de processo consideravelmente. Além disso, ensaios de planejamento fatorial, superfície de resposta e escalonamento em biorreator de bancada indicaram que concentrações baixas de HBC (5-15%, v/v) a 35 °C resultaram na maior produção de BLIS

The great challenge in the food, pharmaceutical and agricultural industries is the search for new compounds to replace antibiotics. Bacteriocins are possible candidates that can be used in parallel with the antibiotics or even to replace them. Lactic-acid bacteria can produce bacteriocin inhibitory substances (BLIS - bacteriocin-like inhibitory substances) that have a bacteriostatic or bactericidal effect against different bacterial species and are widely used as food bioconservatives. In this context, the aim of this work was to use of an agroindustrial waste, hydrolyzed sugarcane bagasse, as a carbon source in fermentative cultures for the production of BLIS by Pediococcus pentosaceus ET 34 strain. Results revealed that ET34 cells were able to grow using this agroindustrial residue as a carbon source, and trials using factorial design showed that agitation did not influence on the production of BLIS. When it was perform cultivation scale up in bioreactors, it was verified that both the growth and the antimicrobial activity were similar to those obtained in the workbench with the exception of the condition of 25% (v/v) of HBC (sugarcane bagasse hydrolyzate) that due to its higher viscosity, resulted in a decrease in growth and BLIS production. BLIS produced by ET34 cells using HBC as a carbon source that was partially purified by ammonium sulfate showed activity against Listeria monocytogenes ATCC 934 and Salmonella enterica CECT 724. Thus, it can be concluded that Pediococcus pentosaceus ET34 was able to grow in medium containing HBC as carbon source producing BLIS in its supernatant with action against different pathogenic bacteria. The possibility of using an alternative carbon source can greatly reduce the process cost. In addition, factorial design, response surface and scale up trials in bench bioreactors indicated that low concentrations of HBC (5-15% v/v) at 35 ºC resulted in higher BLIS production

Optimization of the yield of bacteriocin-like substance (BLIS) produced by Pediococcus pentosaceus and its application as food bioconservative / Otimização do rendimento de substância semelhante a bacteriocina (BLIS) produzido por Pediococcus pentosaceus e sua aplicação como bioconservante de alimentos

Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria (LABs), which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. However, when this bacteriocin has not been completely characterized regarding its amino acid and the nucleotide sequences of the corresponding gene, the qualified term bacteriocin-like inhibitory substance (BLIS) is recommended. In order to increase the antimicrobial activity of bacteriocins, the ability of probiotics LABs, such as Pediococcus pentosaceus, to ferment different carbon and nitrogen sources has been studied. For the development of an improved culture medium, carbon and nitrogen sources must be considered as nutrients responsible for cell growth and bacteriocin production. The best condition, after 48 h of cultivation, for growth (3.420 g/L) and for BLIS production by Pediococcus pentosaceus ATCC 43200 was in Man, Rogosa and Sharp (MRS) culture medium supplemented with 1.5% peptone, initial pH 6.0 and under the following culture conditions: anaerobiosis, 30oC and agitation of 200 rpm. Compared with control (MRS without supplement), the growth of Pediococcus was significantly lower (1.995 g/L) as well as it reduced significantly its generation time from 2.05 h (control) to 1.28 h (MRS supplemented), a reduction of approximately 62.5%. Moreover, addiction of peptone to MRS medium promoted reduction of 4 h to the Pediococcus exponential phase onset. Regarding BLIS antimicrobial activity, addition of nitrogen source to MRS medium was also quite significant. Through the agar diffusion method, BLIS showed inhibition halos between 12.50 and 19.50 mm against LABs strains (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 and Carnobacterium piscicola CECT 4020). Against Listeria strains (Listeria innocua NCTC 111288 and Listeria seeligeri NCTC 11289), their antimicrobial activity was better detected in liquid medium assay, evaluating the minimal inhibitory concentration of 50%. BLIS was able to inhibit 60 and 100% of L. seeligeri and L. innocua, respectively, as well as, diluted 1x (v/v) in water was able to inhibit 100% growth of both Listeria. BLIS 17 showed also good results as food preservative when applied in ready-to-eat pork ham artificially contaminated with L. seeligeri in vacuum-package at 4oC during shelf life of 10 days. BLIS was able to maintain low Listeria multiplication, lower samples weight loss, low lipid peroxidation and good color parameters during samples storage. Results demonstrated the importance of optimizing the culture medium to increase microbial mass, to produce and to improve the activity of this antimicrobial molecule. Moreover, results also suggest the possible application of BLIS as a natural food preservative

Bacteriocinas são peptídeos produzidos por várias espécies de bactérias, especialmente bactérias ácido-láticas (BALs) e apresentam um amplo espectro de ação contra bactérias deteriorantes e patógenos de origem alimentar. Entretanto, quando estas bacteriocinas não foram completamente caracterizadas quanto a sequência de seus nucleotídeos e do seu gene correspondente, é recomendada a denominação de substância semelhante a bacteriocina (BLIS). Para aumentar a atividade antimicrobiana de bacteriocinas, a habilidade de BALs probióticas, como Pediococcus pentosaceus, em fermentar diferentes fontes de carbono e nitrogênio tem sido estudado. Para o desenvolvimento de um meio de cultura melhorado, fontes de carbono e nitrogênio devem ser consideradas como nutrientes responsáveis pelo crescimento celular e pela produção de bacteriocina. A melhor condição, após 48 h de cultivo, para o crescimento (3,420 g/L) e para a produção de BLIS por P. pentosaceus ATCC 43200 foi em meio de cultivo Man, Rogosa e Sharp (MRS) suplementado com 1,5% de peptona, pH inicial 6,0 e sob as seguintes condições de cultivo: anaerobiose, 30oC e agitação de 200 rpm. Comparado ao controle (MRS sem suplementação), o crescimento de Pediococcus foi significativamente menor (1,995 g/L) assim, como também, reduziu significativamente o tempo de geração de 2,05 h (controle) para 1,28 h (MRS suplementado), uma redução de aproximadamente 62,5%. Além disso, a adição de peptona ao meio MRS promoveu redução de 4 h para o início da fase exponencial de Pediococcus. Quanto a atividade antimicrobiana de BLIS, a adição de fonte de nitrogênio ao meio MRS também foi bastante significativa. Através do método ágar difusão, BLIS apresentou halos de inibição entre 12,50 a 19,50 mm contra cepas de BALs (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 e Carnobacterium piscicola CECT 4020). Contra cepas de Listeria (Listeria innocua NCTC 11288 e Listeria seeligeri NCTC 11289), a sua atividade inibitória foi melhor detectada em meio líquido, através da determinação da concentração mínima inibitória de 50%. BLIS sem diluição foi capaz de inibir 60 e 100% de L. seeligeri e L. innocua, 15 respectivamente, assim como, diluído 1x (v/v) em água foi capaz de inibir 100% o crescimento de ambas Listeria. BLIS também apresentou bons resultados como conservante de alimento quando aplicado em presunto contaminado artificialmente com L. seeligeri e armazenado a 4oC a vácuo por 10 dias. BLIS foi capaz de manter baixa a multiplicação de Listeria, menor perda de peso das amostras, baixa peroxidação lipídica e bons parâmetros de cor durante o armazenamento das amostras. Os resultados demonstraram a importância de se otimizar meio de cultivo tanto para o aumento da massa microbiana como para a produção e melhoramento da atividade desta molécula antimicrobiana. Além disso, os resultados também sugerem a possível aplicação de BLIS como conservante natural de alimentos