In vitro evaluation of Lactobacillus gasseri strains of infant origin on adhesion and aggregation of specific pathogens

Numerous Lactobacillus species are members of the normal healthy human intestinal microbiota, and members of the Lactobacillus family predominate among the current marketed probiotic strains. Most of the current commercial probiotic strains have not been selected for specific applications but rather have been chosen based on their technological properties. Often the ability of such strains to temporarily colonize the gastrointestinal tract may be lacking, and the interactions with intestinal microbiota are few. Furthermore, the competitive exclusion properties of potential probiotic bacteria are strain specific and vary greatly. Thus, it is highly desirable that new candidate probiotic isolates originate from the healthy target population. In this study, seven newly isolated strains of Lactobacillus gasseri originating from feces of a healthy newborn child were evaluated for their ability to adhere to intestinal mucus, to autoaggregate and coaggregate with the model pathogens Cronobacter sakazakii (ATCC 29544) and Clostridium difficile (1296). All the bacterial strains, single or in combination, in viable and nonviable forms, were able to autoaggregate. The coaggregation with C. sakazakii or C. difficile was higher (P < 0.05) in nonviable than in the viable forms. Single L. gasseri strains showed similar adhesion abilities to intestinal colon mucus. The seven L. gasseri strains when combined were also able to significantly compete with, displace, and inhibit the adhesion of C. sakazakii and C. difficile in the mucus model. This study demonstrates that the studied L. gasseri strains fulfill the basic adhesion and aggregation properties for probiotics and could be considered for potential future use in children.     

Colicin concentrations inhibit growth of Escherichia coli O157:H7 in vitro

Escherichia coli O157:H7 is a virulent foodborne pathogen that causes severe human illness and inhabits the intestinal tract of food animals. Colicins are antimicrobial proteins produced by E. coli strains that inhibit or kill other E. coli. In the present Study, the efficacy of three pore-forming colicins (El, N, and A) were quantified in vitro against E. coli O157:H7 strains 86-24 and 933. Colicins E1 and N reduced the growth of E. coli O157:H7 strains, but the efficacy of each colicin varied among strains. Colicin E1 was more effective against both strains of E. coli O157:H7 than colicins A and N and reduced (P < 0.05) populations of E. coli O157:H7 at concentrations <0.1 microg/ml. These potent antimicrobial proteins may potentially provide an effective and environmentally sound preharvest strategy to reduce E. coli O157:H7 in food animals.     

Survival and in vivo adhesion of human isolates Lactobacillus gasseri LF221 and K7 in weaned piglets and their effects on coliforms, clostridia and lactobacilli viable counts in faeces and mucosa

In in vivo study on 24 weaned piglets (8 per group), the survival rates of human isolates Lactobacillus gasseri K7 and LF221 were quantified by selective enumeration on MRS agar with rifampicin, and the presence of both strains in intestinal mucosa was examined. Faeces from individual animals were analysed for the number (cfu/g) of coliforms, lactobacilli, clostridia and both of the two probiotic strains during 2-weeks probiotic application period (5 x 10(10) cfu of individual strain/day) and 1 week after the probiotic treatment was ceased. Samples of duodenum, jejunum and ileum of sacrificed animals (5th or 20th day) were also examined microbiologically. A great variability in the microflora of faeces and mucosa was observed even between equally treated animals. The survival of both Lb. gasseri strains was established by their detection in the faeces (2.5 x 10(5) to 3.3 x 10(5) cfu of K7 strain/g faeces; 4.5 x 10(5) to 5 x 10(5) cfu of LF221 strain/g). In two animals, the LF221 or K7 viable cells were found in the faeces 6 d after ceasing probiotic application. In both animals from the group fed with Lb. gasseri K7 that were sacrificed 5 d after weaning, the presence of K7 strain was found either in the mucosa of duodenum (140 cfu/10 cm2) and jejunum (170 cfu/10 cm2) or in the ileum (1600 cfu/10 cm2). LF221 cells were detected in the ileal mucosa of one piglet (820 cfu/10 cm2). The results demonstrated the capability of both tested strains of in vivo adhesion to intestinal mucosa and of temporary colonisation of the piglets' intestine.     

Extracts of Haematoxylon brasiletto inhibit growth, verotoxin production, and adhesion of enterohemorrhagic Escherichia coli O157:H7 to HeLa cells

The extracts of 33 plants were evaluated for their effects on growth of Escherichia coli O157:H7 (EHEC). The extract of Haematoxylon brasiletto was the only one that effectively inhibited bacterial growth. The effects of ethanolic extracts of this plant on growth, verotoxin production, and adhesion of E. coli O157:H7 to HeLa cells were determined. The MBC for growth was 4 mg/ml. No verotoxin formation was detected at 1, 2, or 3 mg/ml. Preexposing bacteria and HeLa cells to various concentrations of extracts affected the adhesion between non-EHEC and HeLa cells. Partial purification of the active fraction suggested that polyphenols might play a role in the antimicrobial activity exhibited by H. brasiletto extracts.     

Probiotic strains: survival under simulated gastrointestinal conditions,in vitro adhesion to Caco-2 cells and effect on cytokine secretion

This study evaluated three probiotic strains (Lactobacillus paracasei subsp. paracasei LC-01, L. acidophilus LA-5, Bifidobacterium lactis Bb-12) and two yoghurt strains (L. delbrueckii subsp. bulgaricus LBY-27 and Streptococcus thermophilus STY-31) with regard to their resistance to simulated gastrointestinal stress, and their ability to interact with human intestinal epithelial cells. The viability of strains was analyzed by measurements of fluorescence-stained cells and their growth by plate colony-counts. The results reveal that for all tested strains, gastric emptying (above pH 3.0) would release a large number of viable cells ranging from 91% for L. paracasei to 53% for S. thermophilus into the intestinal tract, and that between 12 and 23% of them subsequently survive intestinal stress. Among them L. paracasei showed the highest resistance to gastric stress. All the bacteria adhered to the Caco-2 cell line, with the highest adhesions being observed for L. delbrueckii subsp. bulgaricus (9%) and L. acidophilus (7%). Binding of all strains to Caco-2 cells did not result in a significant increase in the production of IL-6 and IL-8 cytokines, suggesting that these bacteria do not trigger an overt inflammatory response in human intestine epithelial cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of tannins on the in vitro growth of Escherichia coli O157:H7 and in vivo growth of generic Escherichia coli excreted from steers

The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement of optical density at 600 nm during anaerobic culture in tryptic soy broth at 37 degrees C, was reduced (P < 0.05) with as little as 400 microg of either tannin extract per ml of culture fluid. The addition of 200, 400, 600, 800, and 1,200 microg of tannins per ml significantly (P < 0.01) reduced the specific bacterial growth rate when compared with the nontannin control. The specific growth rate decreased with increasing dose levels up to 800 microg of tannins per ml. Bacterial growth inhibition effects in chestnut tannins were less pronounced than in mimosa tannins. Chestnut tannin extract addition ranged from 0 to 1,200 microg/ml, and a linear effect (P < 0.05) was observed in cultures incubated for 6 h against the recovery of viable cells, determined via the plating of each strain onto MacConkey agar, of E. coli O157:H7 strains 933 and 86-24, but not against strain 6058. Similar tests with mimosa tannin extract showed a linear effect (P < 0.05) against the recovery of E. coli O157:H7 strain 933 only. The bactericidal effect observed in cultures incubated for 24 h with the tannin preparations was similar, although it was less than that observed from cultures incubated for 6 h. When chestnut tannins (15 g of tannins per day) were infused intraruminally to steers fed a Bermuda grass hay diet in experiment 2, fecal E. coli shedding was lower on days 3 (P < 0.03), 12 (P = 0.08), and 15 (P < 0.001) when compared with animals that were fed a similar diet without tannin supplementation. It was concluded that dietary levels and sources of tannins potentially reduce the shedding of E. coli from the gastrointestinal tract.     

Caseinoglycomacropeptide inhibits adhesion of pathogenic Escherichia coli strains to human cells in culture

Caseinoglycomacropeptide (CGMP) derived from κ-casein was investigated for its ability to inhibit the adhesion of 3 strains of verotoxigenic Escherichia coli (VTEC) and 3 strains of enteropathogenic Escherichia coli (EPEC) to human HT29 tissue cell cultures. Effects on adhesion of Desulfovibrio desulfuricans, Lactobacillus pentosus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus gasseri were also investigated. Generally, CGMP exerted effective anti-adhesive properties at a dose of 2.5 mg/mL, albeit with a high degree of strain specificity. The CGMP reduced adhesion of VTEC strains to <50% of the control and reduced adhesion of EPEC strains to between 80 and 10% of the control. The CGMP also reduced the adhesion of L. pentosus and L. casei to 44 and 42%, respectively. A slight but significant reduction of L. acidophilus, to 81%, was observed, but no significant effects were detected with either Dsv. desulfuricans or L. gasseri. Further investigation of the dose response relationships with the E. coli strains gave IC50 values ranging between 0.12 and 1.06 mg/mL.     

大肠杆菌及乳酸杆菌的辐射致死机理初探

大肠杆菌及乳酸杆菌经过不同剂量的60Co-γ射线的照射,通过计算细菌致死率和DNA漏出率,以及结合透射电镜从微观的方式考察两种菌的致死效应。结果表明,射线对菌体细胞膜的损伤、DNA等胞内蛋白物质的流出是大肠杆菌及乳酸杆菌辐射致死主要的方式。     

嗜酸乳杆菌细菌素Lactobacillin XH2抑制大肠杆菌作用机理的探讨

将离子交换色谱纯化获得的抑菌效价为380.60 AU/mL的嗜酸乳杆菌细菌素Lactobacillin XH2作用于大肠杆菌（Escherichia coli）,从死亡率、细胞膜损伤及引起胞内物质外泄方面,探讨Lactobacillin XH2抑制大肠杆菌的机理。结果表明：Lactobacillin XH2作用于大肠杆菌后,在3 h内大肠杆菌死亡率达96%,菌体细胞变形并出现孔洞,菌体短小且不规整。进一步分析发现,细胞膜通透性增大,胞内K+快速大量泄露,膜电位紊乱失衡;胞内紫外吸收物质、乳酸脱氢酶等生命大分子物质大量流失;同时导致了大肠杆菌胞内三磷酸腺苷（adenosine triphosphate,ATP）外泄,细胞物质代谢、能量代谢失常,最终导致了菌体死亡。这些现象表明,孔道形成（pore formation）理论可能同样适用于Lactobacillin XH2对革兰氏阴性菌大肠杆菌的作用机理。     

Aflatoxin B1 binding by a mixture of Lactobacillus and Propionibacterium: in vitro versus ex vivo

Aflatoxin B1 (AFB) is a well-known carcinogen and reducing its bioavailability is of great interest for human and animal health. Several probiotic bacteria are able to bind AFB1 in vitro, including Lactobacillus rhamnosus LC-705 and Propionibacterium freudenreichii subsp. shermanii JS. A mixture of these two probiotics is used by the food and feed industry as biopreservative (Bioprofit), making it a promising candidate for future applications. Consequently, this study aims to investigate the in vitro and ex vivo ability of this probiotic mixture to bind AFB1. For in vitro experiments, probiotic mixture was suspended in an AFB1 solution (5 microM), incubated for 1 to 30 min, centrifuged, and AFB1 residues were quantitated in supernatant and pellet. For ex vivo experiments, duodenal loops of chicks were ligated and injected with either AFB1 solution alone or probiotic mixture suspension and AFB1 solution. Lumen content was centrifuged and AFB1 was quantitated in supernatant and pellet. Additionally, AFB1 was extracted from duodenal tissue to calculate tissue uptake. In vitro, 57 to 66% of AFB1 was removed from the solution by the probiotic mixture, but only 38 to 47% could be extracted from the bacterial surface. In ex vivo experiments, only up to 25% of AFB1 was bound by bacteria, and tissue uptake of AFB1 was significantly reduced when probiotic bacteria were present in the duodenal loop. Furthermore, the effect of intestinal mucus on the bacterial binding ability was investigated in vitro and was found to significantly reduce AFB1 binding by the probiotic mixture. However, probiotic mixture could only retard but not prevent AFB1 absorption in duodenal loops. Further work needs to assess the potential of probiotics in different experimental setups.     

植物乳杆菌表面性质及对Caco-2细胞的黏附

通过对10 株不同来源的植物乳杆菌进行自聚集能力、表面疏水性以及体外黏附Caco-2细胞能力的测定,探究植物乳杆菌表面性质与黏附能力之间的关系,利用LiCl对植物乳杆菌进行处理,探究参与植物乳杆菌对细胞黏附过程的物质。结果表明：植物乳杆菌3-4对Caco-2细胞的黏附能力最强。所选的不同植物乳杆菌之间自聚集能力、表面疏水性以及对Caco-2细胞的黏附能力存在差异性;对细胞的黏附能力与表面疏水性存在着显著的相关性（P＜0.05）,因此,自聚集能力和疏水性可以作为筛选具有高黏附细胞能力的植物乳杆菌的参考指标。同时LiCl处理前后,植物乳杆菌自聚集能力和对细胞的黏附能力均有所下降,表明菌株表面蛋白类物质及其他大分子物质均参与自聚集和黏附过程。     

High-throughput small molecule screening reveals structurally diverse compounds that inhibit the growth of Escherichia coli O157:H7 in vitro

Escherichia coli O157:H7 colonizes the gastrointestinal tract of ruminants asymptomatically and may enter the human food supply through fecal contamination. A fraction of individuals infected by E. coli O157:H7 develop hemolytic uremic syndrome, a life-threatening condition. When individuals infected by E. coli O157:H7 are treated with certain antibiotics, an increased incidence of hemolytic uremic syndrome may result. This finding supports the need to identify novel compounds that can either reduce the load of E. coli O157:H7 entering the human food supply or serve as alternative therapeutic treatments for infected individuals. We developed a high-throughput turbidometric assay to identify novel compounds that inhibit E. coli O157:H7 growth. Pin transfers were performed to introduce small molecule libraries into 384-well plates, where each well contained approximately 5.0 log CFU of E. coli O157:H7. Plates were incubated at 37°C for 18 h, and the optical density was measured to determine the effect of each small molecule. A total of 64,562 compounds were screened in duplicate, and 43 unique compounds inhibited E. coli O157:H7 growth. Thirty-eight of the 43 inhibitory compounds belonged to known bioactive libraries, and the other 5 compounds were from commercial libraries derived from splitting and pooling. Inhibitory compounds from known bioactive libraries were most frequently therapeutic antibiotics (n = 34) but also included an antiviral compound, a compound that disrupts the citric acid cycle, and two biguanide compounds, which have been used for various nonclinical applications. We identified two novel compounds (i.e., biguanides) that should be studied further for their ability to reduce pathogen populations in foods.     

Effect of xylitol on adhesion of Salmonella Typhimurium and Escherichia coli O157:H7 to beef carcass surfaces

Effects of 10% xylitol (a five-carbon sugar alcohol) on adhesion of Escherichia coli O157:H7 and Salmonella Typhimurium to meat surfaces were examined with three approaches. First, beef outside round was inoculated with rifampin-resistant E. coli O157:H7 and Salmonella Typhimurium dispersed in xylitol or peptone solution. Samples were rinsed with water or not rinsed in a 2 x 2 factorial arrangement. No interaction existed between inoculum and rinsing treatments (P > 0.84). Incubation in xylitol had minimal impact on pathogen adhesion (P > 0.76); however, rinsing reduced pathogen cell counts (P < 0.01). Second, meat samples were treated with water, xylitol, or no rinse; inoculated with pathogens dispersed in peptone solution (8.6 log CFU/ml for each pathogen); and then treated with water, xylitol, or no rinse in a 3 x 3 factorial arrangement. No interactions were observed (P > 0.50). Postinoculation rinsing reduced pathogen loads (P < 0.01) without difference between water and xylitol (P > 0.64). Third, carcass surfaces inoculated with pathogens (5.5 log CFU/cm2) were treated with 35 degrees C water wash, 2.5% L-lactic acid spray, 10% xylitol spray, lactic acid plus xylitol, or hot water plus xylitol. Lactic acid treatments reduced Salmonella Typhimurium at 0 h (P < 0.01) and 24 h (P < 0.02). Hot water treatments tended to reduce Salmonella Typhimurium at 0 h (P < 0.07). Xylitol did not reduce pathogens (P > 0.62) or increase effectiveness of other treatments. Xylitol does not influence E. coli O157:H7 and Salmonella Typhimurium adhesion to meat surfaces.     

乳酸菌代谢产物对大肠杆菌和金黄色葡萄球菌抑制作用的研究

从燕糖酸奶中分离;“乳酸蔺,研究热、酸、pH值处理等对乳酸菌代谢产物抑制大肠杆菌和金黄色葡萄球菌效果的影响。结果表明：乳酸菌发酵液的最终pH值为3．9;乳酸菌代谢产物具有良好的热稳定性以及较好的抗蛋白酶活性和抗过氧化氧酶活性;pH值处删对乳酸菌代谢产物的抑菌能力具有明显影响,pH值越低乳酸菌代谢产物的抑菌能力越强。     

Inhibition of enterotoxigenic Escherichia coli K88, K99 and 987P by the Lactobacillus isolates from porcine intestine

Thirty‐six Lactobacillus strains were isolated from the small intestine of piglets and 14 of them were chosen as they had a good ability (>5 bacteria per cell) to attach to the epithelial cells of the small intestine. They were selected to investigate the antagonistic effects against enterotoxigenic Escherichia coli (ETEC) including K88ab, K88ac, K88+MB (a local strain of haemolytic (H) ETEC K88+ bacterium from Manitoba, Canada), K99 and 987P. Their effects on the adhesion of E coli K88+MB and K88ac to the intestinal mucus of piglets were also studied. The results indicated that six out of the 14 Lactobacillus isolates were able to moderately or strongly inhibit the growth of eight strains of ETEC through the production of organic acids, but not by bacteriocin or hydrogen peroxide. Well diffusion assays of the major organic acids of the Lactobacillus culture supernatants showed that both acetic acid and lactic acid inhibited the growth of E coli. Lactobacillus isolates did not affect the attachment of E coli K88+MB and K88ac to the small intestinal mucus of piglet. © 2000 Society of Chemical Industry     

Binding of aflatoxin B1 alters the adhesion properties of Lactobacillus rhamnosus strain GG in a Caco-2 model

Lactic acid bacteria have been previously reported to possess antimycotoxigenic activities both in vitro and in vivo. The objective of this study was to investigate the effect of aflatoxin B1 on adhesion capability of Lactobacillus rhamnosus strain GG using a Caco-2 adhesion model. Removal of aflatoxin B1 by L. rhamnosus strain GG reduced the adhesion capability of this strain from 30% to 5%. It is therefore concluded that aflatoxins may influence the adhesion properties of probiotics able to sequester them, and subsequently these bacteria may reduce the accumulation of aflatoxins in the intestine via increased excretion of an aflatoxin-bacteria complex.     

Lactobacillus effects on cholesterol: in vitro and in vivo results

A double blind investigation was conducted on the influence of a commercially available tablet containing Lactobacillus acidophilus and Lactobacillus bulgaricus (Lactinex Becton Dickinson Microbiology Systems, Cockeysville, MD) on human serum lipoprotein concentrations. Tablets containing about 2 X 10(6) viable bacteria of Lactobacillus mixtures or placebo tablets were ingested by 354 nonfasting informed subjects in a dose of one tablet each, taken four times a day. There was a 3-wk washout period between two 6-wk treatment periods. The number of viable lactobacillus in unused returned tablets was the same at the end of the study as in the beginning. Analysis of paired data using Wilcoxon signed ranks test showed no major effects on lipoprotein concentrations for either the placebo-treated group or the lactobacilli-treated group. There were no statistically significant differences for low density lipoprotein concentrations between the lactobacilli-treated group and the placebo-treated group. The high density lipoprotein concentrations increased 1.8 to 3.0 mg/dl in both groups for both study periods. For total cholesterol the placebo-treated group experienced a statistically significant increase in the first period according to the Wilcoxon signed ranks test (from 208.0 to 215.0 mg/dl, P less than .001) but not according to a two-sample Student t test. Total cholesterol did not change significantly for the Lactobacillus-treated group in either period. Cardiac risk factor (ratio of total cholesterol to high density cholesterol) did not vary during the study. Lipoprotein values increased immediately following vigorous exercise compared with following 15 min of resting without either placebo or treatment. Sample controls for assay and reassay gave virtually identical values (coefficient of variation 1.6%), confirming that assay results were quite reliable. Thus, ingestion of commercially available Lactobacillus tablets, which contain about 2 X 10(6) cfu/tablet of L. acidophilus and L. bulgaricus cells in a dose of four tablets daily did not affect serum lipoprotein concentrations.     

Bactericidal effects of Lactobacillus reuteri and allyl isothiocyanate on Escherichia coli O157:H7 in refrigerated ground beef

Two naturally occurring antimicrobial agents were tested in packages of refrigerated ground beef for their ability to reduce the viability of Escherichia coli O157:H7 during storage. Allyl isothiocyanate (AITC) and Lactobacillus reuteri were tested separately and together for their action against a cocktail of five strains of E. coli O157:H7 in ground beef held at 4 degrees C for 25 days. Ground beef prepared from whole, raw inside round beef roasts was inoculated with low (3 log CFU/g) or high (6 log CFU/g) levels of the E. coli O157:H7 mixture. The beef was treated with AITC (about 1,300 ppm), L. reuteri, or both, along with 250 mM of glycerol per kg of meat at two levels (3 and 6 log CFU/g) and according to a design that yielded 8 controls plus 10 different treatments. Samples were analyzed for E. coli O157:H7 survivors, numbers of total bacteria, and lactic acid bacteria on days 0 to 25 at 5-day intervals. L. reuteri at both input levels with glycerol killed E. coli O157:H7 at both inoculated levels before day 20. AITC completely eliminated E. coli O157:H7 at the low-inoculum level (3 log CFU/g) and reduced viability >4.5 log CFU/g at the high-inoculum level (6 log CFU/g) by the end of the storage period. The combination of L. reuteri and AITC did not yield an additive effect against E. coli O157:H7 viability. L. reuteri in the presence of glycerol was highly effective against E. coli O157:H7 in ground beef during refrigerated storage (4 degrees C) in modified atmosphere packages. Sensory testing is planned to evaluate effects of treatments.     

玉竹多糖的体外消化特性及其与乳杆菌和大肠杆菌的相互作用

采用水提醇沉法制备玉竹多糖,对其理化性质进行测定;利用体外模拟消化模型研究玉竹多糖在体外模拟唾液、胃和胃肠道中的消化情况;进一步考察其与乳杆菌（植物乳杆菌,CGMCC NO.1258）和大肠杆菌（大肠埃希氏菌,CGMCC NO.1.1521）的相互作用。结果表明,玉竹多糖本身不含还原糖,水解后主要由甘露糖、半乳糖醛酸、葡萄糖、半乳糖组成,含有2种分子质量分布,分别为1.37×106、6.75×103Da;玉竹多糖在被模拟唾液、胃液、胃肠液消化后,还原糖、单糖组成、分子质量均无明显变化,说明玉竹多糖在体外模拟消化系统不能被分解;但在乳杆菌和大肠杆菌的作用下,玉竹多糖发生了不同程度的降解,且玉竹多糖对乳杆菌的生长起到促进作用,对大肠杆菌的生长起到抑制作用。