Selective method for identification and quantification of Bifidobacterium animalis subspecies lactis BB-12 (BB-12) from the gastrointestinal tract of healthy volunteers ingesting a combination probiotic of BB-12 and Lactobacillus rhamnosus GG.

AIM: To develop a novel validated method for the isolation of Bifidobacterium animalis ssp. lactis BB-12 (BB-12) from faecal specimens and apply it to studies of BB-12 and Lactobacillus rhamnosus GG (LGG) recovered from the healthy human gastrointestinal (GI) tract. METHODS AND RESULTS: A novel method for isolating and enumerating BB-12 was developed based on its morphologic features of growth on tetracycline-containing agar. The method identified BB-12 correctly from spiked stool close to 100% of the time as validated by PCR confirmation of identity, and resulted in 97-104% recovery of BB-12. The method was then applied in a study of the recovery of BB-12 and LGG from the GI tract of healthy humans consuming ProNutrients® Probiotic powder sachet containing BB-12 and LGG. Viable BB-12 and LGG were recovered from stool after 21 days of probiotic ingestion compared to baseline. In contrast, no organisms were recovered 21 days after baseline in the nonsupplemented control group. CONCLUSIONS: We demonstrated recovery of viable BB-12, using a validated novel method specific for the isolation of BB-12, and LGG from the GI tract of healthy humans who consumed the probiotic supplement. SIGNIFICANCE AND IMPACT OF THE STUDY: This method will enable more detailed and specific studies of BB-12 in probiotic supplements, including when in combination with LGG.

Role of Hpn and NixA of Helicobacter pylori in susceptibility and resistance to bismuth and other metal ions.

BACKGROUND: Helicobacter pylori produces Hpn, a 60-amino acid, histidine-rich protein that avidly binds nickel and zinc ions, and NixA, a high-affinity nickel transporter in the cytoplasmic membrane. We tested the hypothesis that Hpn and NixA govern susceptibility to metal ions in H. pylori. MATERIALS AND METHODS: Hpn-negative mutants of four H. pylori strains were constructed by standard allelic exchange techniques to yield isogenic Hpn+/Hpn-deficient pairs. A metal concentration that inhibited growth by 50% (IC50) was calculated for Ni2+, Zn2+, Cu2+, and Co2+ by comparing OD600 of cultures in metal-supplemented and control media. RESULTS: Among all four pairs of isogenic strains, the tolerance for Ni2+ was reduced significantly (p <.001) in the Hpn mutants; the mean IC50 value for wild-type strains was 1.9 mM; for the mutant, it was 0.8 mM. In contrast, growth inhibition by Zn2+ was identical within the fours pairs, as was Cu2+ and Co2+ tolerance in one pair tested. We also found that deletion of the hpn gene increases susceptibility to therapeutic forms of bismuth by testing a mutant and wild-type pair with ranitidine bismuth citrate, bismuth citrate, and four antibiotics. Minimal inhibitory concentrations of ranitidine bismuth citrate dropped from 9.2 to 2.3 microg/ml, and those of bismuth citrate dropped from 7.4 to 3.2 microg/ml (p <.05 for both comparisons), while susceptibility to the antibiotics was unaffected. Disruption of the nixA gene encoding the specific Ni2+ transport protein of H. pylori did not change susceptibility to bismuth. CONCLUSION: We concluded that bacteria lacking Hpn, cultured in vitro, are more susceptible than is the wild type to bismuth and Ni2+.

Regulation of the Shiga-like toxin II operon in Escherichia coli.

Investigations of the regulation of the bacteriophage-encoded Shiga-like toxin II (SLT-II) in Escherichia coli demonstrated that bacteriophages exhibit a regulatory impact on toxin production by two mechanisms. Firstly, replication of the toxin-converting bacteriophages brings about an increase in toxin production due to concomitant multiplication of toxin gene copies. Secondly, an influence of a phage-encoded regulatory molecule was demonstrated by using low-copy-number plasmid pADR-28, carrying a translational gene fusion between the promoter and proximal portion of slt-IIA and the structural gene for bacterial alkaline phosphatase (phoA). PhoA activity, reflecting the slt-II promoter activity, was significantly enhanced in E. coli strains which and been lysogenized with an SLT-I or SLT-II-converting bacteriophage (H-19B or 933W, respectively) or bacteriophage lambda. Both mechanisms are dependent on bacteriophage induction and hence are recA dependent. Moreover, the study revealed that the DNA-binding protein H-NS has a regulatory impact on both bacteriophage-mediated SLT-II synthesis and the activity of the slt-II promoter of plasmid pADR-28. While a slight impact of growth temperature on SLT-II expression was observed, no impact of either osmolarity, pH, oxygen tension, acetates, iron level, or utilized carbon source could be demonstrated.

Unique susceptibility of Helicobacter pylori to simethicone emulsifiers in alimentary therapeutic agents.

Helicobacter pylori is killed in vitro by polyoxyethylene acyl esters and ethers similar to simethicone emulsifiers in therapeutic antifoams. The MBC of these compounds for Helicobacter pylori was less than 20 micrograms/ml, while other gram-negative bacteria were unaffected by much higher concentrations of up to 50 mg/ml.

Globotriaosylceramide, Gb3, is an alternative functional receptor for Shiga-like toxin 2e.

We reexamined the binding specificity of the Shiga-like toxin variant associated with porcine edema disease, SLT2e, which is reported to be more cytotoxic for Vero cells than for HeLa cells, by using receptor-deficient cells and a liposomal insertion system for purified glycolipids. We found that SLT2e preferentially uses globotetraosylceramide as a receptor but can also cause cytotoxicity by using globotriaosylceramide, the SLT2 receptor. We conclude that the differential cytotoxicity of SLT2e on HeLa and Vero cells is a function of both the receptor preference of the toxin and the specific glycolipid content of the target cells being used.

Expression and purification of Shiga-like toxin II B subunits.

Shiga-like toxins (SLTs), which are produced by certain strains of Escherichia coli, are composed of enzymatically active A and B subunit multimers responsible for the toxin's binding. We have previously purified large amounts of the SLT-I B subunit by using a hyperexpression vector in Vibrio cholerae under the control of the trc promoter. In this study we examined various expression vectors to maximize yields of the SLT-II B subunit. The SLT-II B subunit has been expressed by using both the T7 promoter and the tac promoter in E. coli. When expressed from a plasmid containing the structural gene for SLT-II B deleted of the leader sequence, SLT-II B was able to form multimers when cross-linked, although SLT-II B production from this plasmid was unreproducible. SLT-II B expressed in all three systems appeared to form unstable multimers, which did not readily bind to a monoclonal antibody which preferentially recognizes B subunit multimers. SLT-II B expression was not increased by moving any of the plasmids into V. cholerae. Polyclonal antibodies raised to SLT-II B in rabbits recognized B subunit in SLT-II holotoxin yet were poorly neutralizing. SLT-II B was also expressed as a fusion protein with maltose-binding protein and could be cleaved from maltose-binding protein with factor Xa. Although the expression vectors were able to make large amounts of SLT-II B, as determined by Western blotting (immunoblotting), the levels of purified SLT-II B subunit were low compared with those obtained previously for SLT-I B subunit, probably because of instability of the multimeric SLT-II B subunit.

Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or iron-regulated promoter systems.

Shiga-like toxin I (SLT-I) B-subunit expression was examined by using the trc promoter in two different constructs, pSBC32 and pSBC54, in which 710 bp of DNA downstream of the B subunit in pSBC32 was deleted. The trc promoter in pSBC54 was replaced also with the SLT-I iron-regulated promoter to create a third plasmid, pSBC61. SLT-I B-subunit expression was examined from all three plasmids following transfer into Escherichia coli JM105 and the cholera toxin A-subunit gene deletion mutant Vibrio cholerae 0395-N1. The SLT-I B subunit was expressed from all constructs. pSBC61 was regulated by elemental iron and produced equivalent amounts of SLT-I B subunit from both E. coli and V. cholerae. In contrast to the cholera toxin B subunit, virtually all released into the medium, the SLT-I B subunit was predominantly cell associated in the pSBC61 constructs. Both pSBC32 and pSBC54 were inducible with isopropyl-beta-D-thiogalactopyranoside (IPTG) in the E. coli background but not the V. cholerae background; however, when E. coli cultures were allowed to grow for 24 h, the yield of SLT-I B subunit was not increased by IPTG induction. Both pSBC32 and -54 expressed more SLT-I B subunit in the V. cholerae host than in the E. coli host. Scale-up to a 9.9-liter fermentor culture of V. cholerae 0395 N1 (pSBC32) resulted in the isolation of 220 mg of SLT-I B. The purified B subunit was identical, in terms of binding to Vero cells, stoichiometry after chemical cross-linking, and ability to inhibit cytotoxicity of intact Shiga toxin, to native SLT-I B subunit from E. coli O157:H7.

One step high yield affinity purification of shiga-like toxin II variants and quantitation using enzyme linked immunosorbent assays.

We have previously purified both shiga-like toxin (SLT) I and II using the toxins' affinity to P1 glycoprotein (P1gp) from hydatid cyst material (HCM). Binding of these toxins is based on their affinity for terminal Gal alpha 1-->4Gal disaccharide residues present in HCM. Although the binding specificity of SLT-II variants (v) differs from that of STL-II they are reported to recognize Gb3 and should bind to P1gp. Therefore we examined the usefulness of HCM to purify SLT-IIv of porcine (p) and human (h) origin. Toxins were purified from fermenter culture supernatants of Escherichia coli HB101 (pDLW5) (SLT-IIvp), and E. coli DH5 alpha (pJES210) (SLT-IIvh) utilizing HCM. SLT-IIvh and SLT-IIvp consisted of A and B subunits, as determined by SDS-PAGE. We obtained 0.16 mg SLT-IIvp and 0.12 mg SLT-IIvh/I of culture (yields > 65%). Various capture systems to detect shiga toxin, SLT-II, SLT-IIvp and SLT-IIvh by ELISA were examined. All toxins bound to HCM, and all except SLT-IIvp bound to the monoclonal antibody 4D1. Only SLT-IIvp bound to the glycolipid Gb4, and only shiga toxin bound significantly to Gb3. Similarities in the level of Gb4 expression in HeLa 229 (ATCC) and Vero cells may explain the lack of differential cytotoxicity between SLT-IIvp and SLT-IIvh on these cell lines.

In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency.

A method for obtaining large numbers of Giardia lamblia cysts in vitro was developed based on modification of earlier methods of in vitro encystation. Maximal numbers of cysts were obtained by growing trophozoites to confluence in TYI-S-33 growth medium containing 0.5 mg/ml of bovine bile, followed by incubation in medium containing 10 mg/ml of bovine bile, at pH 7.8 for 96 h at 37 C. Up to 4 x 10(5) cysts were obtained per milliliter of encystation medium. Cysts thus obtained were similar in structure to those in vivo, were resistant to hypotonic lysis, and reacted with a cyst-specific monoclonal antibody. Further modification of this method by returning the trophozoites to growth medium after 24 hr of exposure to encystation medium resulted in production of cysts that were shown to be viable by fluorogenic dye staining and ability to excyst. This method was scaled up using roller bottles, which resulted in production of up to 1.6 x 10(8) cysts per roller bottle. In addition, of 4 strains tested, the LT strain yielded the highest number of cysts. Of 4 clones of the WB strain, clone A consistently produced the largest number of cysts.

Pseudomonas aeruginosa exoenzyme S requires a eukaryotic protein for ADP-ribosyltransferase activity.

Pseudomonas aeruginosa exoenzyme S ADP-ribosylates several GTP-binding proteins of apparent Mr = 23,000-25,000. Exoenzyme S absolutely requires a soluble eukaryotic protein, which we have named FAS (Factor Activating exoenzyme S), in order to ADP-ribosylate all substrates. The rate of ADP-ribosylation of all exoenzyme S substrates increases linearly with time and with the FAS concentration. FAS is wide-spread in eukaryotes but appears to be absent from prokaryotes. We have estimated the molecular mass of the protein to be approximately 29,000 daltons and its pI to be 4.3-4.5. Several bacterial toxins share this sort of requirement for the presence of a eukaryotic protein for enzymic activity. In particular, FAS resembles ADP-ribosylation factor, a 21,000-dalton GTP-binding protein which performs an analogous function for cholera toxin. However, we can find no evidence that FAS binds GTP. In the presence of FAS, exoenzyme S ADP-ribosylates several proteins in lysates of P. aeruginosa. The requirement for a eukaryotic protein for enzymic activity, which is common to several bacterial toxins, may be a device to identify the eukaryotic environment and to ensure that the enzymes cannot function within and harm the toxin-producing bacteria.

Identification of developmentally regulated Giardia lamblia cyst antigens using GCSA-1, a cyst-specific monoclonal antibody.

GCSA-1, a monoclonal antibody raised against cysts generated in vitro was shown to be Giardia cyst-specific by immunoblot analysis and immunofluorescence. GCSA-1 recognized four polypeptides ranging from 29-45 kD present in the cyst wall. These antigens appeared within eight hours of exposure of trophozoites to encystation medium and were shown to be synthesized by encysting parasites by means of metabolic labelling with [35S]-cysteine. Trophozoites were not stained by the antibody. GCSA-1 also reacted with in vivo cysts obtained from faeces of infected humans, gerbils and mice. These data demonstrate that the determinants recognized by GCSA-1 are early cyst antigens which are developmentally regulated and conserved components of the cyst wall. The actual role of the antigens detected by GCSA-1 in encystation are unknown, but they represent a potential target for strategies directed at inhibiting this process.

Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies.

Shiga toxin from Shigella dysenteriae 60R was purified to homogeneity by a novel one-step receptor analog affinity chromatography method. The method was based on the binding affinity of Shiga toxin for a specific disaccharide, Gal alpha 1----4Gal, which was also present in glycoproteins with P1 blood group seroreactivity produced in hydatid cysts from sheep infected with Echinococcus granulosus. Having shown that cyst fluid P1 glycoprotein bound Shiga toxin on a solid phase, a P1 glycoprotein affinity column was made by coupling P1-active substance to Sepharose 4B. Shiga toxin was purified by this method in large quantities (5 to 10 mg/20-liter batch) with a consistently good yield (greater than 80% of starting toxin). Shiga-like toxins I and II (SLT-I and -II, respectively) from Escherichia coli were also purified by the same method. A preparation containing SLT-II and SLT-I purified by receptor analog affinity chromatography was used to raise four monoclonal antibodies (MAbs) that were reactive with SLT-II by enzyme-linked immunosorbent assay. Three of these antibodies also reacted with Shiga toxin, which was the first clear demonstration of cross-reactivity between these toxins. One MAb, 4D1, which was specific for the B subunit of SLT-II and Shiga toxin, neutralized both toxins in a HeLa cell cytotoxicity assay. Two MAbs recognized the A subunit of both SLT-II and Shiga toxin by Western blot (immunoblot) analysis but were unable to neutralize either toxin. In addition, one B-subunit-specific MAb neutralized SLT-II alone, and a previously described Shiga toxin B-subunit-specific MAb was shown to be specific for Shiga toxin but not SLT-II.

Identification and characterization of taglin, a mannose 6-phosphate binding, trypsin-activated lectin from Giardia lamblia.

We have previously reported the presence of a cell surface associated lectin activity in Giardia lamblia, a human protozoan parasite that is a significant cause of diarrheal disease worldwide [Lev, B., Ward, H., Keusch, G. T., & Pereira, M. E. A. (1986) Science (Washington, D.C.) 232, 71-73]. This lectin is specifically activated in vitro by a host protease, trypsin, which is secreted in vivo at the site of infection. The activated lectin agglutinates cells to which the parasite adheres in vivo and binds specifically to isolated brush border membranes of these cells. These findings suggest that this lectin may be of importance in the host-parasite interaction. We now report the identification of this lectin, which we have named taglin (to denote trypsin-activated Giardia lectin), and describe some of its properties. A monoclonal antibody that inhibits the hemagglutinating activity of taglin recognizes a protein of 28,000/30,000 kdaltons in Western blots of Giardia lysates. This finding was confirmed by direct demonstration of lectin activity with the technique of erythrocyte binding to proteins electroblotted to nitrocellulose, which revealed specific red cell binding to giardial protein bands in the same molecular weight range as those recognized by the monoclonal antibody. This study also elucidates the binding of taglin to terminal phosphomannosyl residues. The involvement of cell surface phosphate in binding of taglin to erythrocytes is shown by the abolition of lectin activity by alkaline phosphatase treatment of the erythrocytes. Taglin also requires divalent cations, Ca2+ or Mn2+, for hemagglutinating activity and is active within a narrow pH range of 6-7.