LACTOBACILLUS PLANTARUM ISOLATED FROM KEFIR GRAINS AND ITS ANTAGONIC EFFECTS AGAINST SHIGELLA sp. INVASION

KAKISU E.; BOLLA, PATRICIA ARACELI; FERNANDEZ M; DE URRAZA P.J; DE ANTONI G.L

Cordoba

Congreso; SAMIGE; 2009

Shigella sp. is one of the most important causes of acute diarrhoea in developing countries. In our country the most frequent species identified are S. flexneri and S. sonnei. Shigella sp. is the etiological agent of a disease known as shigellosis or human dysentery. The early essential step to cause shigellosis is the invasion of colonic epithelial cells, followed by intracellular bacterial multiplication and spread of invading bacteria into adjacent epithelial cells. In vitro models have been used to study cell invasion and subsequent events that allow efficient bacterial colonization of the epithelial layer It is known the use of probiotics to prevent gastrointestinal disorders, including infectious diseases caused by enteric pathogens. In our laboratory has been reported the ability of some bacteria isolated from kefir grains to antagonized different enteropathogenic bacteria such as Salmonella and enterohemorragic Escherichia coli. For this study we selected two enteroinvasive Shigella strains, obtained from clinical cases: Shigella flexneri 72 and Shigella sonnei 45. Three potentially probiotic microorganisms were used to study their inhibitory activity against Shigella: Lactobacillus plantarum (CIDCA 83114), Kluyveromices marxianus (CIDCA 8154) and Streptococcus thermophilus (CP2 strain). Invasion of Hep-2 cells was studied with and without previous incubation of Shigella with Lb. plantarum and the mixture of lactic acid bacteria and yeast. Both, Shigella and inhibitory microorganisms at concentration of 108 CFU/well were added to Hep-2 monolayer and incubated during one hour. Similar experiments were performed by pre-incubating bacterial walls obtained from Lb. plantarum. Enumeration of Shigella was performed by plate-count on nutrient agar. Unlike the control in which the pathogen had invaded the Hep-2 cells, the results showed a significant reduction of Shigella invasion when cells were treated with lactobacilli and the mixture of potentially probiotic strains. In order to explore the protection mechanisms, co-aggregative properties of strains and the action of proteolytic enzymes were tested. Co-aggregation was determined by measuring optical density. The results obtained indicate that protection against Shigella could not be attributed to co-aggregation with lactic acid bacteria or yeast since any OD reduction or aggregation of microorganisms has been detected. An anti-invasive effect has been observed with Lb. plantarum bacterial walls. Treatment of Lb. plantarum with pepsin decreased the antagonic effect against Shigella. The results obtained demonstrate that certain protection effects against invasion may be ascribed to bacterial surfaces. In conclusion, we have found that Lb. plantarum isolated from kefir lactobacilli has the ability to protect Hep-2 cells against invasion by Shigella flexneri and Shigella sonnei. This property could be associated to the presence of lactobacilli surface proteins.