Browsing by Author "Borges, Sandra Cristina Ferreira"
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- Selection of an antimicrobial culture to be used in the prevention of neonatal listeriosisPublication . Borges, Sandra Cristina Ferreira; Teixeira, Paula Cristina Maia; Silva, Joana Gabriela LaranjeiraThe capacity of lactic acid bacteria to produce acidic products and/or secrete antimicrobial compounds is important in the impairment of vaginal colonization by pathogens. Vaginal pH is normally acidic, varying between 3.5-4.5; an increase in vaginal pH (5.0 to 6.5) can be associated with colonization by pathogenic microorganisms. The main goal of this study was to select an antimicrobial culture to be used in the prevention of vaginal colonization of Listeria monocytogenes during pregnancy, and consequently, prevent neonatal listeriosis. The survival and biofilm formation of 20 isolates of L. monocytogenes in simulated vaginal fluid at normal vaginal pH (4.2) and at higher pH values (5.5 and 6.5) was investigated. This pathogen was inhibited by the normal vaginal pH but survives when pH increases. All isolates tested were biofilm producers at different pH values. Streptococcus agalactiae is an important cause of neonatal infection and maternal colonization. Therefore, its behavior in simulated vaginal fluid was also analyzed. As with L. monocytogenes, S. agalactiae (n=10) survived longer at higher pH values than at normal vaginal pH. All S. agalactiae isolates were also biofilm producers. Therefore, since L. monocytogenes and S. agalactiae can survive at higher vaginal pHs, fetuses/neonates from women having increased vaginal pH values during pregnancy, may be at higher risk of neonatal infection. Biofilm production increases the probability of occurrence of neonatal infection. The application of vaginal probiotics could have the potential for preventing vaginal Listeria colonization in pregnant women and consequently reduce neonatal infections. Thirty-five isolates of Pediococcus spp. showed antimicrobial activity against L. monocytogenes, by production of a bacteriocin, but did not inhibit S. agalactiae isolates. Pediococcus spp. isolates demonstrated the ability to survive in simulated vaginal fluid at pH 4.2. Based on the higher bacteriocinogenic activity and survival in simulated vaginal fluid, one isolate of Pediococcus spp. was selected and characterized to evaluate its safety before use as vaginal probiotic. Pediococcus pentosaceus SB83 did not show the presence of virulence factors such as the production of gelatinase, lipase and DNase, hemolytic activity, nor the presence of virulence genes (i.e. surface adhesin, aggregation protein, cytolysin and extracellular metallo-endopeptidase). No relevant antibiotic resistance traits were detected. Pediococcus pentosaceus SB83 produced biofilms at different pH values (4.2, 5.5 and 6.5) in simulated vaginal fluid, which could also serve as a protective layer against colonization by pathogenic bacteria. The bacteriocin produced by P. pentosaceus SB83, designated as bacteriocin SB83, also showed inhibitory activity against Enterococcus faecalis and Enterococcus faecium, but did not inhibit vaginal lactic acid bacteria. Bacteriocin SB83 is resistant to several conditions, including conditions in the vaginal tract (pH and components of vaginal fluid). The bacteriocinogenic activity of P. pentosaceus SB83 against L. monocytogenes was evaluated in simulated vaginal fluid at pH 6.5, since this is the ideal pH to the L. monocytogenes survival and proliferation. There the inhibitory effect of the bacteriocinogenic culture was assessed in suspension, as lyophilized powder and in tablets. Suspensions of P. pentosaceus SB83 (1010 CFU/mL) reduced the pathogen (10 CFU/mL) 5after only 2 h of exposure to below the detection limit; the lyophilized bacteria after 24 h of contact and in tablet form, P. pentosaceus SB83 lost the antimicrobial activity. The pH of simulated vaginal fluid decreased in all the tested conditions. Since P. pentosaceus SB83 lose its antimicrobial activity in tablet form, it could be therefore used in the form of lyophilized powder, which may be administered intra-vaginally, for instance as a washing solution. This formulation was selected to evaluate the anti-listerial activity during 12 months of storage. During storage at room temperature, lyophilized bacteria totally inhibited the pathogen (below the detection limit) only during one month; after this time, there was a decrease in the cell counts of P.pentosaceus SB83 and, consequently, in antimicrobial potential. During storage at 4 ºC, P. pentosaceus SB83 showed antimicrobial activity throughout the time of storage investigated. The bacteriocin produced by P. pentosaceus SB83 after storage at 4 ºC, remained active at least during 12 months, however a slight decrease in antimicrobial activity occurred between 9 to 12 months. Therefore, the best formulation of P. pentosaceus SB83 is as a lyophilized powder stored at 4 ºC. These in vitro results prove a concept for the use of P. pentosaceus SB83 as a vaginal probiotic, to prevent vaginal colonization by L. monocytogenes in pregnant women.