Immunomodulating properties of various microbes--representatives of normal intestinal microflora]

The impact of oral administration of heat-killed bifidobacteria, lactobacteria, enterococci and bacteroides on mouse resistance to experimental salmonellosis and content of immunoglobulin-synthesizing cells in the proper plate of the small intestine was studied. It was shown that the administration of the killed bifidobacteria, lactobacteria and enterococci increased the animal resistance to experimental salmonellosis infection and induced an increase in the content of immunoglobulin-synthesizing cells in the proper plate of the small intestine. The administration of the killed bacteroides had no such effect. Possible development of bacterial preparations with immunomodulating properties based on killed bifidobacteria, lactobacteria or enterococci is discussed.     

Isolation of human faecal bifidobacteria which reduce signs of Salmonella infection when orogastrically dosed to mice

AIMS: The aim of the study was to isolate human bifidobacteria that inhibit growth of Salmonella typhimurium in vitro, and provide protection against Salmonella infection in mice. METHODS AND RESULTS: A total of 92 micro-organisms, which displayed antagonist activity against Salm. typhimurium in vitro, were isolated from human faecal material. Based on their Gram stain status, cultures were pooled and tested for anti-Salmonella activity. The Gram-variable group was the most active. From that group, three bifidobacteria (Laftitrade markB22, B74 and B97) individually showed good pathogen inhibition in vivo. CONCLUSION: Oral administration of certain human bifidobacteria provides protection against Salmonella infection in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that certain bifidobacteria may be used as a prophylaxis for reduced incidence and severity of Salmonella infections.     

Correction of intestinal microflora in chemotherapeutic dysbacteriosis using bifidobacterial and lactobacterial autologous strains

The oral administration of kanamycin (40 mg/kg) or ampiox (500 mg/kg) to guinea pigs for 5 days led to disturbances in their normal intestinal microflora, manifested by a sharp decrease in the levels of lactobacteria and bifidobacteria, as well as by the appearance of large amounts of enterobacteria and enterococci, normally not detected in the proximal and distal sections of the intestinal tract. In adult volunteers receiving kanamycin orally in a dose of 40 mg/kg for 5 days disturbances in microbiocenosis also occurred: the amount of enterococci, staphylococci, lactobacteria and bifidobacteria considerably decreased, enterobacteria becoming the dominating microorganisms. Three oral administrations of bifidobacterial and lactobacterial autostrains immediately after the abolition of the antibiotic facilitated the rapid and effective restoration of the intestinal microflora.     

Increased resistance of mice to Salmonella enterica serovar Typhimurium infection by synbiotic administration of Bifidobacteria and transgalactosylated oligosaccharides

AIMS: The anti-infectious activity of Bifidobacteria in combination with transgalactosylated oligosaccharides (TOS) against Salmonella enterica serovar Typhimurium LT-2 in an opportunistic antibiotic-induced murine infection model in mice was examined. METHODS AND RESULTS: B. breve (strain Yakult) with natural resistance to streptomycin sulphate (SM, MIC: > 4 mg ml(-1)), when given daily at a dose of 108 cfu/mouse orally under SM treatment was constantly excreted at 10(10) cfu g(-1) faeces so long as SM was administered, even at 2 weeks after discontinuing administration of B. breve. Explosive intestinal growth and subsequent extra-intestinal translocation of orally infected LT-2 under SM treatment were inhibited by B. breve colonization, and this anti-infectious activity was strengthened by synbiotic administration of TOS with B. breve. Comparison of anti-Salmonella activity among several Bifidobacterium strains with natural resistance to SM revealed that strains such as B. bifidum ATCC 15696 and B. catenulatum ATCC 27539T conferred no activity, even when they reached high population levels similar those of effective strains such as strain Yakult and B. pseudocatenulatum DSM 20439. Both the increase in the concentration of organic acids and the lowered pH in the intestine due to bifidobacterial colonization correlated with the anti-infectious activity. Moreover, the crude cecal extract of B. breve-colonized mice exerted growth-inhibitory activity against LT-2 in vitro, whereas that of the ineffective B. bifidum-colonized cecum showed much lower activity. CONCLUSIONS: Intestinal colonization by bifidobacteria given exogenously together with TOS during antibiotic treatment prevents the antibiotic-induced disruption of colonization resistance to oral infection with S. enterica serovar Typhimurium, and the metabolic activity needed to produce organic acids and lower the intestinal pH is important in the anti-infectious activity of synbiotics against enteric infection with Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that certain bifidobacteria together with prebiotics may be used for the prophylaxis against opportunistic intestinal infections with antibiotic-resistant pathogens.     

The effect of enterococci on normalizing the intestinal microflora in experimental dysbacteriosis

The normal microflora of the intestine produces essential influence on the vital activity of the host. The exposure of the body to the action of different unfavorable factors (roentgen radiation, the administration of antibiotics, Salmonella infection, etc) results in changes in the normal microflora of the gastrointestinal tract. This work was aimed at the study of the influence of Streptococcus faecium YDC-48 on the intestinal microflora of mice in experimental (chemotherapeutic, postirradiation) dysbacteriosis and Salmonella infection. The effect of the oral administration of S. faecium YDC-48 on the correction of the intestinal microflora of mice in cases of dysbacteriosis etiology was studied. The intragastric administration of S. faecium YDC-48 was found to induce an increase in the level of lactobacterin and a decrease in the number of opportunistic microorganisms in chemotherapeutic and postirradiation dysbacteriosis. The oral administration of S. faecium YDC-48 decreased the manifestations of intestinal dysbacteriosis in experimental Salmonella infection. The possibility of developing a preparation on the basis of S. faecium YDC-48, a representative of normal intestinal microflora, is discussed.     

Immunomodulation by Yersinia enterocolitica: comparison of live and heat-killed bacteria

This study compared the immunomodulating properties of viable and killed Yersinia enterocolitica O9 in BALB/c mice. At 10 days after infection by the intragastric route, ex vivo assays showed a suppression of spleen cell proliferation in response to Salmonella lipopolysaccharide, concanavalin A and heat-killed yersiniae. Mice infected with Y. enterocolitica O9 for 10 days resisted the challenge with a lethal dose of Listeria monocytogenes. In contrast, intravenous administration of heat-killed yersiniae did not modify the ability of spleen cells to proliferate in response to lipopolysaccharide or concanavalin A, and proliferation in response to killed yersiniae was significantly increased. By 3 days after administration of a single dose of heat-killed yersiniae, the resistance of mice to L. monocytogenes challenge was significantly increased. Our findings show profound differences in immunomodulation by viable and heat-killed yersiniae, but suggest that killed yersiniae retain interesting immunomodulating properties.     

Effects of Indomethacin on Acute, Subacute, and Latent Infections in Mice and Rats

The comparative effect of indomethacin and hydrocortisone on the resistance of mice or rats to various acute, subacute, and latent bacterial infections was investigated. Large daily doses of indomethacin and hydrocortisone administered to mice challenged with bacterial pathogens, including Klebsiella pneumoniae AD, Salmonella schottmuelleri 3010, Staphylococcus aureus (Smith), Streptococcus pyogenes C203, Salmonella pullorum #2, Proteus vulgaris 1810, and Pseudomonas aeruginosa 2616, revealed that in essentially all of these acute infections, the mortality of the infected mice treated with indomethacin was essentially identical to that found in the infected controls. In contrast, hydrocortisone often lowered the resistance of mice to these acute infections. In a more chronic bacterial infection due to Corynebacterium kutscheri, hydrocortisone produced striking deleterious effects on resistance, whereas indomethacin administration in doses approaching the maximal tolerated level caused no observable adverse effects on host resistance. Indomethacin fed continuously to rats for 80 days, at maximal tolerated levels, caused no observable adverse effects on the host-parasite relationship of rats which were shown to harbor various latent infections. Hydrocortisone administration, however, lowered the resistance of rats as evidenced by increased mortality related directly to extensive bacterial infection. Insofar as infection is concerned, indomethacin behaved like other nonsteroid anti-inflammatory agents such as aspirin and phenylbutazone.     

Cytophilic immunoglobulins on the surface of neutrophilic granulocytes in mice perorally immunized with a live vaccine made from the supressor revertant S. typhimurium Rev 8]

The reaction of the spleen cell migration inhibition in the presence of monospecific antisera against mouse G, A and M immunoglobulins was used to detect cytophilic antibodies on the surface of mouse granulocytes. The oral administration of ACR live vaccine from suppressor revertant Salmonella typhimurium Rev. 8 protected the mice against infection induced by virulent species of mouse. Salmonella typhimurium. The immunized mice showed an increase in cytophilic IgG on the surface of neutrophile granulocytes.     

Isolation of a Soluble Resistance-Enhancing Factor from Mycobacterium phlei

Fox, Alfred E. (Warner-Lambert Research Institute, Morris Plains, N.J.), Joachim Anschel, George L. Evans, Raam R. Mohan, and Benjamin S. Schwartz. Isolation of a soluble resistance-enhancing factor from Mycobacterium phlei. J. Bacteriol. 92:285-290. 1966.-Extraction of a crude cell wall preparation from Mycobacterium phlei with 20% urea yielded a fraction which induced a state of enhanced resistance to microbial challenge. The resulting soluble extract, after removal of the urea, represented a 15% yield of solids with the separation of the biologically active component(s) and elimination of toxicity. Single oral or subcutaneous submicrogram doses of this material induced a prolonged state of increased resistance to subsequent challenge with Salmonella enteritidis in mice. This effect appeared as early as 2 hr after oral administration and persisted for at least 30 days. Protection against experimental infection with Staphylococcus aureus was also demonstrated. Resistance to viral challenge with influenza type A was observed after intranasal administration of the M. phlei extract to mice. The isolated material was found to contain carbohydrate, protein, nucleic acids, and lipids. The lipids represented 60% of the total solids, and were all short-chain fatty acids. No toxic effects, including pyrogenicity, could be demonstrated after oral or parenteral administration of this preparation.     

In vivo-selected mutations in methyl-directed mismatch repair suppress the virulence attenuation of Salmonella dam mutant strains following intraperitoneal, but not oral, infection of naïve mice

Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-AP(r)) isolates in systemic tissues of mice infected with dam mutant Salmonella. Such 2-AP(r) derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of na?ve mice. These 2-AP(r) derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.     

Effect of Streptococcus faecalis BIO-4R on intestinal flora of weanling piglets and calves

The effect of oral administration of Streptococcus faecalis BIO-4R, an antibiotic-resistant lactic acid bacterium, on the intestinal flora of weanling piglets and cows reared on antibiotic-containing diet was investigated. Fourteen days after administration of the bacteria, the intestinal flora of the piglets was examined. Animals of the administered group had stabilized lactic flora such as bifidobacteria, streptococci, and lactobacilli, whereas most animals of control group had reduced lactic flora. On the other hand, abundant yeasts were detected from the cecum, colon, and feces of the control animals, but the levels were significantly lower in the animals given strain BIO-4R. The density of Salmonella in the intestine appeared to be reduced after the administration of strain BIO-4R. The number of BIO-4R cells was shown to be 10 times lower in the duodenum and jejunum than in the ileum, suggesting that strain BIO-4R might have grown transiently in the ileum. The similar trend toward stabilization of the lactic flora was also observed in cows after administration of BIO-4R. In addition, an antagonistic effect of the strain against yeasts and Salmonella was suggested. These findings indicate that the oral administration of strain BIO-4R is one of the useful methods whereby the potentially deleterious effect of antibiotics on the intestinal flora of farm animals may be minimized.     

Experimental Salmonellosis VIII. Postinfective Immunity and Its Significance for Conferring Cellular Immunity

In the process of live-vaccine immunization of Salmonella enteritidis infection in mice, the relation between the number of bacteria in the organs of mice and their protecting effect was studied. Treatment with antibiotics was used to control the number of immunizing bacteria in the tissues. Mice, which were infected with 10(-5) mg (1,000 mouse MLD) of virulent S. enteritidis and treated with kanamycin simultaneously, acquired high antilethal resistance against infection with the same organisms. However, the administration of large amounts of kanamycin, which caused a rapid decrease in bacterial numbers in the organs of infected mice, was incapable of conferring immunity. This indicated the necessity of persistence of live bacteria in the host for the production of immunity. A large number of microorganisms were maintained for 53 weeks in a diffusion chamber inserted into the mouse abdominal cavity. The mice implanted with diffusion chambers containing large numbers of virulent S. enteritidis did not acquire antilethal resistance against infection with the same organisms, although agglutinins against S. enteritidis were observed in these mice. Agglutinin was also found in the fluid contained in diffusion chambers inserted into mice immunized with a killed vaccine of S. enteritidis. This indicated that antibody penetrated the membrane filter of diffusion chambers from outside to inside and vice versa. From these results, it is suggested that contact of live microorganisms with the host cell is necessary for conferring postinfective immunity in salmonellosis.     

Effect of Streptococcus faecalis BIO-4R on intestinal flora of weanling piglets and calves.

The effect of oral administration of Streptococcus faecalis BIO-4R, an antibiotic-resistant lactic acid bacterium, on the intestinal flora of weanling piglets and cows reared on antibiotic-containing diet was investigated. Fourteen days after administration of the bacteria, the intestinal flora of the piglets was examined. Animals of the administered group had stabilized lactic flora such as bifidobacteria, streptococci, and lactobacilli, whereas most animals of control group had reduced lactic flora. On the other hand, abundant yeasts were detected from the cecum, colon, and feces of the control animals, but the levels were significantly lower in the animals given strain BIO-4R. The density of Salmonella in the intestine appeared to be reduced after the administration of strain BIO-4R. The number of BIO-4R cells was shown to be 10 times lower in the duodenum and jejunum than in the ileum, suggesting that strain BIO-4R might have grown transiently in the ileum. The similar trend toward stabilization of the lactic flora was also observed in cows after administration of BIO-4R. In addition, an antagonistic effect of the strain against yeasts and Salmonella was suggested. These findings indicate that the oral administration of strain BIO-4R is one of the useful methods whereby the potentially deleterious effect of antibiotics on the intestinal flora of farm animals may be minimized.     

双歧杆菌的生物拮抗与药敏试验

本文介绍了双歧杆菌生物拮抗试验和药物敏感试验的方法。结果说明双歧杆菌对肠道正常菌群及病原菌有调节与拮抗作用,且对多种抗生素敏感。提示在治疗肠道感染时,应尽量避免口服对双歧杆菌敏感的药物,否则会使肠道感染加重或蔓延。     

四君子汤对实验性脾虚小鼠肠道菌群的影响

本文报道了四君子汤对脾虚小鼠肠道内与人类关系密切的主要菌群的影响。用大量大黄水煎液灌胃给予小鼠,造成实验性脾虚模型,引起小鼠肠道内菌群紊乱,其中双歧杆菌、乳杆菌菌量均下降,与对照组小鼠相比具有显著性差异。当小鼠服用四君子汤后,双歧杆菌,乳杆菌均上升至正常值水平。四君子汤对拟杆菌数量也有一定的影响。本研究表明,四君子汤对小鼠肠道菌群的失调具有调节功能,对脾虚小鼠的康复具有一定的促进作用。     

双歧杆菌对门静脉血内毒素影响初步研究

本实验以大鼠为动物模型,探讨在肠道微生态失调的状态下,用大量双歧杆菌(Bifidobacteria)灌服,借以拮抗外源性需氧革兰氏阴性杆菌的生长繁殖,从而使肠源性内毒素自门静脉的吸收减少。对正常鼠、脱污染鼠、再污染鼠、治疗鼠和非治疗鼠,进行不同肠段的双歧杆菌、需氧革兰氏阴性杆菌的测定和门静脉血内毒素测定。结果表明:双歧杆菌对肠道需氧革兰氏阴性杆菌的生长繁殖,具有明显的生物拮抗作用,同时伴有相应的门静脉血内毒素水平的降低。     

Oral administration of lactobacilli from human intestinal tract protects mice against influenza virus infection

Aims: Our study was conducted to evaluate the potent protective effects of oral administration of probiotic Lactobacillus strains against influenza virus (Flu) infection in a mouse model. Method and Results: Lyophilized Lactobacillus rhamnosus GG (LGG) and Lactobacillus gasseri TMC0356 (TMC0356) were orally administered to BALB/c mice for 19 days. The test mice were intranasally infected with Flu A/PR/8/34 (H1N1) on day 14, and any changes in clinical symptoms were monitored. After 6 days of infection, the mice were killed and pulmonary virus titres were determined. The clinical symptom scores of mice administered oral LGG and TMC0356 were significantly ameliorated, compared to those of the control mice (P < 0·01). The pulmonary virus titres of the mice fed LGG and TMC0356 were also significantly decreased compared to those of control mice (P < 0·05). Conclusions: These results indicate that oral administration of lactobacilli, such as LGG and TMC0356, might protect a host animal against Flu infection. Significance and Impact of the Study: These results demonstrate that oral administration of selected lactobacilli might protect host animals from Flu infection by interactions with gut immunity.     

Stimulation of nonspecific resistance to infection by a crude cell wall preparation from Mycobacterium phlei

Fox, Alfred E. (Warner-Lambert Research Institute, Morris Plains, N.J.), George L. Evans, Frank J. Turner, Benjamin S. Schwartz, and Ansel Blaustein. Stimulation of nonspecific resistance to infection by a crude cell wall preparation from Myocobacterium phlei. J. Bacteriol. 92:1-5. 1966.-Exposure of large quantities of viable Mycobacterium phlei to attrition in a colloid mill resulted in 90 to 95% disruption of the organisms. Isolation of the crude cell wall preparation was accomplished by centrifugation of the broken cells at 10,000 x g, resuspension of the sediment, and repeated centrifugation at 1,000 x g to remove intact cells. Single oral or parenteral doses of the cell wall preparation increased the resistance of mice and guinea pigs to experimental infection with Salmonella enteritidis, and of mice to Staphylococcus aureus, for prolonged periods after administration. Histological examination of the organs of mice treated orally or intraperitoneally revealed a lymphoid hyperplasia of the spleen and a Kupffer cell proliferation of the liver. The preparation was nontoxic to mice by the oral route at doses up to 5,000 mg/kg, and the intraperitoneal ld(50) was approximately 680 mg/kg.     

Low-dose Salmonella infection evades activation of flagellin-specific CD4 T cells

Many pathogens can establish a lethal infection from relatively small inocula, yet the effect of infectious dose upon CD4 T cell activation is not clearly understood. This issue was examined by tracking Salmonella flagellin-specific SM1 T cells in vivo, after i.v. and oral challenge of mice with virulent Salmonella typhimurium. SM1 T cells rapidly expressed activation markers and expanded in response to high-dose infection but remained completely unresponsive in mice challenged with low doses of Salmonella. SM1 T cells, in these mice, remained unresponsive, despite massive bacterial replication in vivo. Naive SM1 T cells in low-dose Salmonella-infected mice were activated rapidly after the injection of flagellin peptide, demonstrating that these T cells were fully capable of responding, ruling out the possibility of a bacterial-induced suppressive environment. The inability of flagellin-specific SM1 T cells to respond to low-dose infection was not due to Ag down-regulation, because flagellin expression was detected using a functional assay. Together, these data suggest that low-dose Salmonella infection can evade flagellin-specific CD4 T cell activation in vivo.     

Fasciola hepatica: attempts to induce protection against infection in rats and mice by injection of excretory/secretory products of immature worms

In vitro excretory/secretory products of 4-week (immature) and 8-week-old (mature) Fasciola hepatica parasites, derived from rats, were injected together with adjuvant into naive rats and mice. Resistance to infection was assessed in rats by counting adults in the bile ducts at 9 weeks, or in mice by recording deaths after oral challenge with a high dose of viable metacercariae. Exposure of rats to excretory/secretory products of immature F. hepatica conferred a significant degree of resistance which was comparable to the level of resistance induced following oral administration of a low number of metacercariae. No protection against infection was seen in rats injected with excretory/secretory products from mature, bile duct-derived worms. In mice, no obvious mouse strain variation in susceptibility to first infection existed and hypothymic nude mice were as susceptible to infection as intact mice. As determined by protection against death, vaccination with excretory/secretory products derived from immature F. hepatica was without effect in mice. It is concluded that "host protective antigens", at least for rats, were present in the excretory/secretory products of immature F. hepatica larvae.