In Vitro and In Vivo Inhibition of Helicobacter pylori by Lactobacillus casei Strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

Antagonistic Activity against Helicobacter Infection In Vitro and In Vivo by the Human Lactobacillus acidophilus Strain LB

The purpose of the present study was to examine the activity of the human Lactobacillus acidophilus strain LB, which secretes an antibacterial substance(s) against Helicobacter pylori in vitro and in vivo. The spent culture supernatant (SCS) of the strain LB (LB-SCS) dramatically decreased the viability of H. pylori in vitro independent of pH and lactic acid levels. Adhesion of H. pylori to the cultured human mucosecreting HT29-MTX cells decreased in parallel with the viability of H. pylori. In conventional mice, oral treatment with the LB-SCS protected against infection with Helicobacter felis. Indeed, at both 8 and 49 days post-LB-SCS treatment (29 and 70 days postinfection), inhibition of stomach colonization by H. felis was observed, and no evidence of gastric histopathological lesions was found. LB-SCS treatment inhibits the H. pylori urease activity in vitro and in H. pylori that remained associated with the cultured human mucosecreting HT29-MTX cells. Moreover, a decrease in urease activity was detected in the stomach of the mice infected with H. felis and treated with LB-SCS.     

Evaluation of the antagonistic effect of Lactobacillus acidophilus on clinical strains of Helicobacter pylori

The purpose of the study was to determine the antagonistic effect of Lactobacillus strains, isolated from saliva of healthy people on clinical strains of H. pylori. Two kinds of Lactobacillus strains were used in the tests i.e. strains producing hydrogen peroxide as well as strains not producing the metabolite. Clinical isolates of H. pylori were taken from the patients with confirmed duodenal and gastric ulcers. The evaluation of antagonistic effect between microorganisms was performed by using agar slab method. It was observed that Lactobacillus acidophilus had an antagonistic effect on growth of all strains of H. pylori tested. However, the particular Lactobacillus strains differed with respect to their inhibitory properties. Hydrogen peroxide did not have any significant impact on the inhibition of the growth of Helicobacter pylori.     

复方嗜酸乳杆菌片联合三联方案根除幽门螺杆菌的临床研究

目的探讨复方嗜酸乳杆菌片联合三联方案根除幽门螺杆菌(Helicobacter pylori,H.pylori)的疗效及不良反应。方法将143例H.pylori检测阳性的患者随机分为A1组(48例)、A2组(46例)和B组(49例)。B组给予三联方案(埃索美拉唑、阿莫西林、克拉霉素)根除H.pylori治疗,疗程10d。A1组、A2组分别在上述三联方案的基础上同时加用10d、20d的复方嗜酸乳杆菌片。治疗过程中观察并记录上述三组的不良反应发生情况,停药4周后查13 C呼气试验判别H.pylori根除是否成功。结果共133例患者完成治疗和随访,A1组、A2组、B组根除率按方案(PP)分析分别为65.9%、69.8%、63.0%,按意向性(ITT)分析分别为60.4%、65.2%、59.2%,无论按方案分析还是意向性分析三组根除率差异均无统计学意义(P0.05)。纳入三组的共143例患者均完成不良反应的随访,A1组、A2组、B组不良反应发生率分别为10.4%、8.7%、30.6%,三组相比差异有统计学意义(P0.05),其中A1组、A2组分别与B组相比差异有统计学意义(P0.05),A1组与A2组相比差异无统计学意义(P0.05)。结论三联方案加用10d、20d的复方嗜酸乳杆菌片未能提高H.pylori根除率,但可降低不良反应发生率。     

Coccoid Helicobacter pylori Not Culturable In Vitro Reverts in Mice

An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the “dormant” forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD₅₅₀ = 5 Mc Farland) of Helicobacter pylori with bacillary (2-5×10⁹ CFU/ml) and coccoid (0 CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.     

Inhibitory effect of Lactobacillus acidophilus on Helicobacter hepaticus in vitro

Helicobacter hepaticus and Helicobacter pylori both belong to Helicobacter species. Strains of Lactobacillus acidophilus, including L4 and L6, have shown significant inhibitory effects on H. pylori. Based on this phenomenon, we aim to investigate the inhibitory effect of L. acidophilus on H. hepaticus. Both standard and isolated H. hepaticus strains were grown under microaerophilic conditions at 37 °C in the presence of L. acidophilus supernatant, or lactic acid. The diameters of the inhibition zones were measured on the solid culture media. In liquid culture, the cell concentrations were measured and the urease activity was determined by phenol red staining. Sixteen strains of L. acidophilus isolated from human feces (named as L1–L16) showed anti-H. hepaticus effects. Two of them (L4 and L6) exhibited the most apparent effects on H. hepaticus inhibition. The L. acidophilus supernatant of L4 and L6 significantly increased the diameters of the inhibition zones compared with that of the lactic acid control (P < 0.05). The inhibitory role of L. acidophilus supernatant was independent of the pH value of solution (P > 0.05). Moreover, in liquid culture, L. acidophilus supernatant significantly reduced the cell growth rate and the urease activity of H. hepaticus cells in a time-dependent pattern (P < 0.05 compared with lactic acid control). No obvious difference was observed between the standard and isolated strain of H. hepaticus (P > 0.05). Our results indicate that L. acidophilus can decrease the viability and urease activity of H. hepaticus in vitro and this inhibition is independent of pH levels. This provides evidence for developing novel approaches for the prevention and treatment of H. hepaticus infection.     

In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

Peptide Extracts from Cultures of Certain Lactobacilli Inhibit Helicobacter pylori

Helicobacter pylori inhibition by probiotic lactobacilli has been observed in vitro and in vivo. Carefully selected probiotic Lactobacillus strains could therefore play an important role in the treatment of H. pylori infection and eradication. However, the underlying mechanism for this inhibition is not clear. The aim of this study was to examine if peptide extracts, containing bacteriocins or other antibacterial peptides, from six Lactobacillus cultures (Lactobacillus acidophilus La1, Lactobacillus amylovorus DCE 471, Lactobacillus casei YIT 9029, Lactobacillus gasseri K7, Lactobacillus johnsonii La1, and Lactobacillus rhamnosus GG) contribute to the inhibition of H. pylori. Peptide extracts from cultures of Lact. amylovorus DCE 471 and Lact. johnsonii La1 were most active, reducing the viability of H. pylori ATCC 43504 with more than 2 log units within 4 h of incubation (P < 0.001). The four other extracts were less or not active. When six clinical isolates of H. pylori were tested for their susceptibility towards five inhibitory peptide extracts, similar observations were made. Again, the peptide extracts from Lact. amylovorus DCE 471 and Lact. johnsonii La1 were the most inhibitory, while the three other extracts resulted in a much lower inhibition of H. pylori. Protease-treated extracts were inactive towards H. pylori, confirming the proteinaceous nature of the inhibitory substance.     

Zymogram and Preliminary Characterization of Lactobacillus helveticus Autolysins

The autolysins of Lactobacillus helveticus ISLC5 were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymogram). By using lyophilized Micrococcus luteus cells or heated whole cells of L. helveticus ISLC5 (0.2% [wt/vol]) as a substrate, several lytic activities were detected in the whole-cell SDS extract of strain ISLC5 (i) one activity at 42.4 kDa, which was named autolysin A, and (ii) six other activities having very similar molecular weights (29.1, 29.6, 30, 30.8, 31.7, and 32.8 kDa), which were named autolysins B (B1 through B6, respectively). As regards the temporal distribution of the enzymes, autolysins A and B were detected in the cells harvested from the beginning of the exponential growth phase. Autolysin A appeared to be associated only with viable cells, whereas the autolysins B remained associated with the cell envelope several days after the complete loss of culture viability. When SDS-treated walls of L. helveticus ISLC5 were used as a substrate, a supplementary lytic activity appeared at 37.5 kDa; it was considered a peptidoglycan hydrolase, since it was not able to induce lysis of whole-cell substrate. The autolysins of 30 other strains of L. helveticus from various geographical origins were also analyzed by zymogram; all the activity profiles obtained were similar to that of strain ISLC5 in terms of the number of lytic bands and their apparent molecular weights. Only the relative intensities of the lytic bands corresponding to autolysins A and B were variable depending on the strains. This observation suggested that autolysins are highly conserved enzymes. A concentrated crude lysate of the virulent bacteriophage 832-B1 infecting L. helveticus was also analyzed by zymogram; one lytic activity with an apparent molecular weight of 31.7 kDa, very close to the weights of the autolysins B, was observed. Finally, the autolysins of L. helveticus ISLC5 were successfully extracted from whole cells by using a 1 M lithium chloride solution; they were partially purified by precipitation, selective resolubilization, and gel filtration chromatography, which led to a 20-fold increase in specific activity.     

In vitro growth inhibition of Helicobacter pylori by lactobacilli belonging to the Lactobacillus plantarum group

AIMS: The aim of this study was to test and locate the in vitro anti-Helicobacter activity of seven Lactobacillus strains belonging to Lactobacillus plantarum group. METHODS AND RESULTS: Growth inhibition of H. pylori was tested using a well-plate assay. Of the strains displaying the strongest growth inhibition, a L. plantarum isolated from sauerkraut (MLBPL1) was chosen for further studies. The detected anti-Helicobacter activity of MLBPL1 was mainly associated with cell wall, and to a minor extent with the culture supernatant. The active component, which was determined to be between 3 and 10 kDa in size, retained its activity after 10 min treatment at 100 degrees C. The activity was present when MLBPL1 was cultivated in rich laboratory cultivation medium MRS and in different food matrices. CONCLUSIONS: The strains belonging to L. plantarum group showed anti-Helicobacter activity in vitro. The main activity seemed to be associated with cell wall rather than culture supernatant or intracellular fraction. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the rapid spread of resistant H. pylori strains caused by antibiotic therapy, addition of a fermented food containing L. plantarum to the conventional antibiotic treatment of Helicobacter infection could establish a potential complementary means to suppress the infection.     

Effect of Lactobacillus acidophilus and Bifidobacterium bifidum supplementation to standard triple therapy on Helicobacter pylori eradication and dynamic changes in intestinal flora

To investigate Lactobacillus acidophilus (L. acidophilus) and Bifidobacterium bifidum (B. bifidum) supplementation to triple therapy for Helicobacter pylori (H. pylori) eradication and dynamic changes in intestinal flora in children with H. pylori infection. One hundred H. pylori-infected children were randomly assigned to two groups: treatment group (n = 43), standard triple anti-H. pylori therapy plus probiotics of L. acidophilus and B. bifidum for 2 weeks followed by taking probiotics for another 4 weeks; control group (n = 45), standard triple anti-H. pylori therapy for 6 weeks. After 6-week treatment, ¹³C-urease breath test was performed and side effects were monitored during the observation period. Quantitative PCR with 16S rRNA-gene-targeted species-specific primers was carried out for the analysis of human intestinal B. bifidum, L. acidophilus, and Escherichia coli (E. coli). As expected, treatment group could significantly enhance the H. pylori eradication rate (83.7 vs. 64.4 %, P < 0.05). B. bifidum, L. acidophilus, and E. coli showed no statistical difference before or after therapy in the treatment group. The number of B. bifidum and L. acidophilus was significantly decreased after 2-week treatment in the control group, but after 6-week treatment it significantly increased and nearly returned to the level before treatment. The number of E. coli increased significantly after 2-week treatment, while after 6-week treatment, it nearly decreased to the level before treatment. L. acidophilus and B. bifidum supplementation is effective for H. pylori eradication compared with triple therapy alone.     

不同干预时机下复方嗜酸乳杆菌片联合四联疗法在根除幽门螺杆菌失败补救治疗中的应用

目的探讨不同干预时机下复方嗜酸乳杆菌片联合四联疗法在首次根除幽门螺杆菌(H. pylori)失败患者补救治疗中的作用。方法选择西安市第一医院消化内科90例经标准四联疗法根除H. pylori失败的患者为研究对象,随机分为研究组A(A组)、研究组B(B组)与对照组(C组),各30例。A组患者使用复方嗜酸乳杆菌片联合补救四联疗法治疗。B组患者在补救四联疗法结束后序贯使用复方嗜酸乳杆菌片2周。C组患者单独采用补救四联疗法治疗。比较3组患者临床症状、不良反应发生率及H. pylori根除率。结果A组、B组、C组患者总有效率分别为93.33%(28/30),90.00%(27/30),66.67%(20/30),差异有统计学意义(χ²=9.120,P=0.010)。A组、B组、C组患者总不良反应发生率分别为13.33%(4/30)、16.67%(5/30)、66.67%(20/30),差异有统计学意义(χ²=24.522,P<0.001)。A组、B组、C组患者H. pylori根除率分别为60.00%(18/30)、63.33%(19/30)、33.33%(10/30),差异有统计学意义(χ² =6.502,P=0.039)。A组与B组患者H. pylori根除率相比差异无统计学意义(χ²=0.071,P=0.791)。结论复方嗜酸乳杆菌片联合四联疗法可提高首次四联疗法根除H. pylori失败患者H. pylori根除率,改善患者临床症状,降低总体不良反应发生率,但不同干预时机下复方嗜酸乳杆菌片对H. pylori根除率无明显影响。     

Lactase Production from Lactobacillus acidophilus

Summary A lactobacillus strain isolated from fermented Ragi (Eleusine coracana Gaertn.) was characterized as Lactobacillus acidophilus. The isolate was found to be homofermentative, slime-forming and a lactase (β-galactosidase) producer. Production, recovery, characterization and performance of lactase were studied at laboratory scale from 100 ml to 5 l under stationary and stirred conditions. 1.5% lactose was found to be the best carbon source for lactase production. The lactose content could be reduced to 0.75% by supplementing with 1% ragi, thus making the media economically more attractive. A 6.5-fold increase (5400 U ml⁻¹) was achieved on scale-up. Performance of the lactase obtained from this strain was found to be slightly better than the commercial lactase produced by Kluyveromyces lactis.     

Protoplast fusion between Lactobacillus casei and Lactobacillus acidophilus

Summary From the fusion between Lactobacillus casei and Lactobacillus acidophilus, 8 fusants were selected: Four were able to ferment maltose, lactose, galactose and mannose, but two had greater abilities of acid production than parents. Increased values of up to 7.6–8 % in -galactosidase activity were obtained from two when compared to that of L. acidophilus, whereas another 2 had activities of 800 and 548 nmol/mg protein/min comparable to that of L casei giving a value of 400 nmol/mg protein/min in phospho--galactosidase activity.     

胃黏膜定植乳酸杆菌对幽门螺杆菌感染的影响

目的探讨胃黏膜定植乳酸杆菌对幽门螺杆菌感染及胃黏膜菌群数量的影响。方法收集130例慢性胃炎患者胃窦黏膜组织,病理学检测幽门螺杆菌,提取胃黏膜基因组DNA,采用荧光定量PCR法检测乳酸杆菌和总细菌数。结果幽门螺杆菌阳性组和阴性组的乳酸杆菌检出率和乳酸杆菌数(Log)差异均无统计学意义(P0.05);乳酸杆菌阳性者和阴性者之间胃黏膜总细菌数(Log)差异无统计学意义(P0.05);乳酸杆菌细菌数(Log)与胃黏膜总细菌数(Log)无显著相关性(P0.05);不同炎症程度胃炎患者乳酸杆菌检出率差异无统计学意义(P0.05);肠化组和未肠化组乳酸杆菌检出率差异无统计学意义(P0.05);但是重度胃炎组幽门螺杆菌感染率和总细菌数量均显著高于轻度(P0.05)和中度胃炎组(P0.05)。结论胃黏膜定植乳酸杆菌对幽门螺杆菌的感染无影响,其存在与否及细菌数量对胃黏膜总细菌数亦无明显影响,并与胃炎炎症程度及肠化无关。乳酸杆菌不能通过抑制幽门螺杆菌定植、调节胃黏膜菌群而减轻炎症反应。     

Streptococcus mitis Induces Conversion of Helicobacter pylori to Coccoid Cells during Co-Culture In Vitro

Helicobacter pylori (H. pylori) is a major gastric pathogen that has been associated with humans for more than 60,000 years. H. pylori causes different gastric diseases including dyspepsia, ulcers and gastric cancers. Disease development depends on several factors including the infecting H. pylori strain, environmental and host factors. Another factor that might influence H. pylori colonization and diseases is the gastric microbiota that was overlooked for long because of the belief that human stomach was a hostile environment that cannot support microbial life. Once established, H. pylori mainly resides in the gastric mucosa and interacts with the resident bacteria. How these interactions impact on H. pylori-caused diseases has been poorly studied in human. In this study, we analyzed the interactions between H. pylori and two bacteria, Streptocccus mitis and Lactobacillus fermentum that are present in the stomach of both healthy and gastric disease human patients. We have found that S. mitis produced and released one or more diffusible factors that induce growth inhibition and coccoid conversion of H. pylori cells. In contrast, both H. pylori and L. fermentum secreted factors that promote survival of S. mitis during the stationary phase of growth. Using a metabolomics approach, we identified compounds that might be responsible for the conversion of H. pylori from spiral to coccoid cells. This study provide evidences that gastric bacteria influences H. pylori physiology and therefore possibly the diseases this bacterium causes.