Identificación molecular de bacterias ácido lácticas con potencial probiótico aislado de heces de neonatos humanos

Abstract

El objetivo de este estudio fue aislar e identificar molecularmente bacterias con potencial probiótico de heces de neonatos humanos y determinar su cinética de crecimiento. Se evaluó 30 muestras de heces de neonatos nacidos vía vaginal y 30 por cesárea (0-3 días). Las muestras se enriquecieron en caldo Man Rogosa Sharp (MRS) a 37°C por 24h. Se sembraron en agar MRS y se contabilizaron colonias GRAM positivas, catalasa negativa y libre de esporas. Se aislaron cepas en agar MRS modificado a 37°C por 24h, de donde se seleccionaron colonias para pruebas in vitro con sales biliares; resistencia a pH bajo y actividad antimicrobiana. Frente a Escherichia coli ATCC25922, E. coli ATCC35218, Salmonella entérica y Listeria inocua mediante ensayos de difusión en agar. La identificación molecular se realizó con amplificaciones BOX-PCR y el secuenciamiento del gen 16S rRNA. Se aislaron 48 cepas y todas presentaron resistencia a pH 3 y 0.3% sales biliares en diferente porcentaje; tres cepas mostraron actividad antimicrobiana a E. coli ATCC25922, 1 cepa a E. coli ATCC35218, 5 cepas a L. inocua y todas a S. entérica. Se obtuvieron dos perfiles BOX-PCR, de los cuales nueve cepas (C52, C61, C71, C112, C16 2, C192, C20, C35, y C42) presentaron 100% de similaridad a Lactobacillus plantarum ATCC 14917T [ACGZ01000098] y dos (C15 y C40) 99.93% y 99.80% de similaridad a Enterococcus faecium CGMCC 1.2136T [AJKH01000109]; estas cepas mostraron actividad en leche con diferencias significativas (p valor < 0.05) en cinética de crecimiento. En conclusión, se logró la identificación de cepas Lactobacillus y Enterococcus con potencial probiótico.

The objective of this study was to isolate and molecularly identify bacteria with probiotic potential from human neonatal feces and characterize their growth kinetics. Thirty stool samples from neonates born vaginally and 30 by caesarean section (0-3 days) were evaluated. The samples were enriched in Man Rogosa Sharp broth (MRS) at 37 ° C for 24h. They were grown on MRS agar and the GRAM positive, catalase negative and spore-free colonies were counted. Strains were isolated on modified MRS agar at 37 ° C / 24h, from which colonies were selected for in vitro tests resistance to bile salts, to low pH and antimicrobial activity against Escherichia coli ATCC25922, E. coli ATCC35218, Salmonella enteric and Listeria innocua by diffusion in agar. The molecular identification was made with PCR-BOX amplifications and the sequencing of the 16S rRNA genes. Forty eight strains were isolated and all showed resistance to pH 3 and 0.3% bile salts in different percentages; three strains showed antimicrobial activity against to E. coli ATCC25922, one strain to E. coli ATCC35218, five strains to L. innocuous and all to S. enterica. Two BOXPCR profiles were obtained, of which nine strains (C52, C61, C71, C112, C162, C192, C20, C35, and C42) had 100% similarity to Lactobacillus plantarum ATCC 14917T [ACGZ01000098] and two (C15 and C40) 99.93% and 99.80% similarity to Enterococcus faecium CGMCC 1.2136T [AJKH01000109]; these strains showed activity in milk with significant differences (p value <0.05) in growth kinetics. In conclusion, Lactobacillus and Enterococcus with probiotic potential were identified.