The influence of glycerol upon L. reuteri activity against enteropathogens / A influência do glicerol sobre a atividade de L. Reuteri contra enteropatógenos

BACKGROUND: Probiotics containing L. reuteri are popular for treating and preventing bacterial gastrointestinal infections. L. reuteri, produces reuterin, an antibiotic that inhibits gram-negative bacteria. Reuterin production is the result of glycerol fermentation by L reuteri. Although L. reuteri is normally present in the gastrointestinal system, only small amounts of glycerol are usually available; therefore, the production of reuterin may not occur and this could reduce the effectiveness of the probiotic supplement. Our objective is to identify the minimum concentrations of glycerol required for L. reuteri to exert an inhibitory effect on enteropathogenic enterobacteriaceae. METHOD: Samples containing 10(8) colony forming units (CFU) of L. reuteri DSM17938 (Colikids®, Ache, Brazil/BioGaia, Sweden) were grown with varying concentrations of glycerol (0.05-5%). 10(6) CFU of E.coli (CDC0126/INCQS/FIOCRUZ), Shigella flexneri (ATCC/120022), S. enterica (ATCC6539) and Y. enterocolitica (ATCC9610) were inoculated with L. reuteri in the different glycerol concentrations. Each enterobacteria and glycerol 5% without L. reuteri cultures were used as positive control groups. RESULTS: All bacteria were completely inhibited at higher ranges of glycerol concentrations (0.2-5%) and grew at lower concentrations (0.05-0.1%). CONCLUSION: L. reuteri requires at least 0.2% of glycerol to completely inhibit enterobacterial growth. These preliminary findings may influence the current method of use of probiotic supplements. The antibiotic activity of L. reuteri may have potential clinical use against important enteropathogens.

CONTEXTO: Os probióticos que contêm L. reuteri são populares para tratar e prevenir infecções bacterianas do trato gastrointestinal. L. reuteri produz reuterina, um antibiótico que inibe bactérias gram-negativas. A produção de reuterina depende da presença de quantidades adequadas de glicerol, cuja fermentação resulta na produção do antibiótico. Embora L. reuteri esteja normalmente presente no sistema GI, apenas pequenas quantidades de glicerol estão geralmente disponíveis; portanto, a produção de reuterina pode não ocorrer o que poderia reduzir a eficácia do suplemento probiótico. Nosso objetivo é identificar as concentrações mínimas de glicerol necessárias para que L. reuteri exerça um efeito inibitório nas enterobacteriaceas enteropatogênicas. MÉTODO: 108 CFU de L. reuteri DSM17938 (Colikids®, Ache, Brasil / BioGaia, Suécia) cresceu com concentrações variáveis de glicerol (0,05-5%). Foram inoculadas 106 UFC de E. coli (CDC0126 / INCQS / FIOCRUZ), Shigella flexneri (ATCC / 120022), S. enterica (ATCC6539) e Y. enterocolitica (ATCC9610) com L. reuteri nas diferentes concentrações de glicerol. Cada enterobacteria e glicerol 5% sem culturas de L. reuteri foram utilizadas como grupos de controle positivo. RESULTADOS: Todas as bactérias foram completamente inibidas em maiores concentrações de glicerol (0.2-5%) e cresceram em concentrações mais baixas (0.05-0.1%). CONCLUSÃO: L. reuteri requer pelo menos 0,2% de glicerol para inibir completamente o crescimento de enterobacteria. Essas descobertas preliminares podem influenciar o método atual de uso de suplementos de probióticos. A atividade antibiótica de L. reuteri pode ter potencial uso clínico contra importantes enteropatógenos.

Las posibilidades del empleo de lactobacilos en la gastroenterología y la nutrición humana / Therapeutic potential of the use of lactobacilli in gastroenterology and human nutrition

Lactobacilli are normal inhabitants of the human intestine. They are also present in maternal milk, where Lactobacillus reuteri was first described. This confirms that maternal milk transfers components of the maternal microbiota to the offspring. L. reuteri ATCC 55730 decreases the intensity and duration of infantile colic defined as episodes of strident crying for at least 3 hours, 3 times a week during 3 months. Its etiology is unknown but recent evidence shows that affected infants harbor less Iactobacilli and more Bacteroides in their feces. In 2008 it was shown that this strain carries plasmidia that could transfer resistance to antibiotics. For this reason L. reuteri it was cured of these factors and this resulted in L. reuteri DSM 17938 which is devoid of them; tests in vitro, in laboratory animals and in humans demonstrated that both strains are equivalent. The effect of L. reuteri in infantile colic may be explained by its blocking at the level of the dorsal ganglia of the spinal chord of the transmission to the central nervous system of afferent pain stimuli. L. reuteri has effects on gastroesophageal reflux probably through the acceleration of gastric emptying and it decreases the incidence and severity of necrotizing enterocolitis. Other positive effects are related to chronic constipation and acute diarrhea. L. reuteri synthesizes a molecule with intense bactericidal activity, β-hidroxy propenal o reuterin from glycerol, which may explain some of its effects. Lactobacillus rhamnosus GG ATCC 53103 is probably the probiotic that has been studied the most and for a longer period. It shortens the duration and it decreases the number of evacuations in acute diarrhea as well as the duration and frequency of vomiting. It also induces improvements of antibiotic associated diarrhea and of upper respiratory infections. The positive effects of Lactobacillus GG may be due to the stimulation of innate and adaptive immunity and to the activation of macrophages and of NK lymphocytes. One of its effects refers the response its administration induces in infants with atopy/eccema. Some studies have demonstrated that the severity and extent of the lesions is decreased after Lactobacillus GG administration, that may persist for some years after its administration has been ended. Lactobacillus GG also exerts positive effects on conditions such as the irritable bowel syndrome through mechanisms that are not known with certainty.

Los lactobacilos son habitantes normales de las mucosas de los seres humanos; también están presentes en la leche materna. Lactobacillus reuteri fue descrito en la leche de una madre peruana, lo que confirma que estas bacterias son parte de la microbiota que las madres transfieren a sus hijos. El cólico infantil son episodios de llanto excesivo, estridente, por a lo menos 3 horas 3 veces por semana y por a lo menos 3 meses. L. reuteri ATCC 55730 demostró disminuir su intensidad y duración. La etiología del cólico se desconoce pero en las heces de los lactantes afectados se han demostrado disminuciones de los lactobacilos y aumentos de Bacteroides. En 2008 se demostró que L. reuteri ATCC 55730 era portador de plasmidios capaces de resistencia a antibióticos por lo que fue "curado" de estos factores y resultó una nueva cepa, denominada DSM 17938; pruebas in vitro y en animales mostraron su inocuidad y ensayos en humanos revelaron que ejerce las mismas propiedades benéficas que la cepa original. El efecto de L. reuteri en el cólico infantil puede estar relacionado con su capacidad de bloquear en los ganglios dorsales de la medula espinal la transmisión de estímulos a los centros cerebrales superiores. El efecto antirreflujo de L. reuteri se debería a la aceleración del vaciamiento gástrico. Ambas cepas de esta bacteria disminuyen la progresión, morbilidad y mortalidad de la enterocolitis necrosante. Otros efectos en la constipación crónica y la duración de la diarrea aguda de distintas etiologías. L. reuteri sintetiza una molécula, el β-hidroxipropenal o reuterina, con actividad antibacteriana. Lactobacillus rhamnosus GG ATCC 53103 es probablemente el probiótico más estudiado; acorta la duración de la diarrea aguda, disminuye el número de evacuaciones y la duración de los vómitos e induce mejorías de la diarrea asociada con los tratamientos antibióticos; y disminuiría la intensidad de las infecciones respiratorias altas. Sus efectos se deberían a la estimulación de las inmunidades innata y adquirida y de la activación de los macrófagos y los linfocitos NK. Un efecto notable y discutido de L. ramnosus GG se refiere a sus efectos en la atopia cutánea, ya que disminuiría la extensión e intensidad de sus síntomas, incluso más allá de su administración. También ejercería efectos sobre cuadros funcionales gastrointestinales como el síndrome del intestino irritable, por mecanismos aun no elucidados.