Characterisation of Yersinia pestis isolates from natural foci of plague in the Republic of Georgia, and their relationship to Y. pestis isolates from other countries.

Forty Yersinia pestis isolates from endemic foci of plague in the Republic of Georgia, and six Y. pestis isolates from neighbouring former Soviet Union countries, were analysed for their biochemical and phenotypic properties, and their genetic relatedness was compared with Y. pestis strains KIM and CO92 by pulsed-field gel electrophoresis (PFGE). In addition, 11 Y. pestis isolates from the USA, together with published nucleotide sequences from Y. pestis strains KIM, CO92 and 91001, were compared with the 46 isolates in the present collection using multilocus sequence typing (MLST), based on sequence data for the 16S rRNA, hsp60, glnA, gyrB, recA, manB, thrA and tmk loci. Four virulence gene loci (caf1, lcrV, psaA and pla) were also sequenced and analysed. Two sequence types (ST1 and ST2), which differed by a single nucleotide, were identified by MLST. With the exception of a single isolate (771G), all of the Georgian Y. pestis isolates belonged to ST2. PFGE also grouped the Georgian Y. pestis isolates separately from the non-Georgian isolates. Overall, PFGE discriminated the Y. pestis isolates more effectively than MLST. The sequences of three of the four virulence genes (lcrV, psaA and pla) were identical in all Georgian and non-Georgian isolates, but the caf1 locus was represented by two allele types, with caf1 NT1 being associated with the non-Georgian isolates and caf1 NT2 being associated with the Georgian isolates. These results suggest that Georgian Y. pestis isolates are of clonal origin.

Improved pulsed-field gel electrophoresis for typing vancomycin-resistant enterococci.

A rapid protocol for subtyping vancomycin-resistant enterococci by pulsed-field gel electrophoresis is reported. The procedure is simple and potentially cost-effective and allows reproducible subtyping of the strains in approximately 1 day.

Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+.

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

Effects of trachynilysin, a protein isolated from stonefish (Synanceia trachynis) venom, on frog atrial heart muscle.

The effects of trachynilysin (TLY), a protein toxin isolated from stonefish (Synanceia trachynis) venom, were studied on the electrical and mechanical activities of frog atrial fibres. TLY (1 microg/ml) hyperpolarized the membrane, shortened the action potential (AP) duration (APD), exerted a negative inotropic effect and elicited contracture. These effects did not develop in the presence of atropine. TLY shortened the APD of fibres isolated from a frog completely paralyzed with botulinum type A toxin, in the presence of Ca2+ but not when Ca2+ was replaced by Sr2+. TLY increased the basal and the peak of the fluorescence ratio of stimulated fibres loaded with fura-2. Confocal laser scanning microscopy revealed the existence of a diffuse innervation in atrial tissue. Our results suggest that TLY enhances the release of acetylcholine from atrial cholinergic nerve terminals and activates indirectly muscarinic receptors leading to a shortening of APD. They also show that the mechanical effects induced by TLY are due to an increase of the Ca2+ influx and to a rise in intracellular Ca2+ levels which leads to (i) a slowing of the Na+/Ca2+ exchange activity, which accounts for the contracture and (ii) the activation of a Ca2+-dependent K+ current involved in the APD shortening.

Production of enterotoxin by Yersinia bercovieri, a recently identified Yersinia enterocolitica-like species.

Yersinia bercovieri, a recently identified Y. enterocolitica-like species, produces a heat-stable enterotoxin (designated YbST) which has biologic activity in infant mice and increases short circuit current in Ussing chambers. Although YbST has some properties in common with the heat-stable enterotoxins of Y. enterocolitica (YST I and YST II), it appears to be a novel toxin because (i) it was not neutralized by anti-YST I antiserum, (ii) YbST-neutralizing antiserum did not neutralize YST I, and (iii) Y. bercovieri strains did not hybridize with genetic probes for yst I, yst II, and other known enterotoxins.

Selective depletion of clear synaptic vesicles and enhanced quantal transmitter release at frog motor nerve endings produced by trachynilysin, a protein toxin isolated from stonefish (Synanceia trachynis) venom.

Our previous observation that low concentrations of stonefish (Synanceia trachynis) venom elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals prompted our present study to purify the quantal transmitter-releasing toxin present in the venom and to characterize the toxin's ability to alter the ultrastructure and immunoreactivity of frog motor nerve terminals. Fractionation of S. trachynis venom by sequential anion exchange fast protein-liquid chromatography (FPLC) and size-exclusion FPLC yielded a highly purified preparation of a membrane-perturbing (haemolytic) protein toxin, named trachynilysin. Trachynilysin (2-20 micrograms/ml) significantly increased spontaneous quantal acetylcholine release from motor endings, as detected by recording miniature endplate potentials from isolated frog cutaneous pectoris neuromuscular preparations. Ultrastructural analysis of nerve terminals in which quantal acetylcholine release was stimulated to exhaustion by 3 h exposure to trachynilysin revealed swelling of nerve terminals and marked depletion of small clear synaptic vesicles. However, trachynilysin did not induce a parallel depletion of large dense-core vesicles. Large dense core vesicles contained calcitonin gene-related peptide (CGRP), as revealed by colloidal gold immunostaining, and trachynilysin-treated nerve endings exhibited CGRP-like immunofluorescence similar to that of untreated terminals. Our results indicate that the ability of stonefish venom to elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals is a function of trachynilysin, which selectively stimulates the release of small clear synaptic vesicles and impairs the recycling of small clear synaptic vesicles but does not affect the release of large dense-core vesicles. Trachynilysin may be a valuable tool for use in other secretory terminals to discriminate between neurotransmitter and neuropeptide release.

Capsular polysaccharide-protein conjugate vaccines of carbotype 1 Vibrio vulnificus: construction, immunogenicity, and protective efficacy in a murine model.

Vibrio vulnificus causes septicemia and wound infections in immunocompromised humans. The capsular polysaccharide of Vibrio vulnificus (VvPS) is critical for virulence. We synthesized conjugate vaccines of carbotype 1 VvPS under conditions and in formulations suitable for human use. Purified VvPS was conjugated to tetanus toxoid (TT) or to inactivated V. vulnificus cytolysin or elastase by two different schemes. All conjugates elicited elevated anticapsular immunoglobulin G (IgG) and IgM and antiprotein IgG responses in mice compared with saline placebo. The conjugates prepared through caboxyl activation of VvPS (VvPS-TTa, VvPS-cytolysin, and VvPS-elastase) were more immunogenic than the one prepared through hydroxyl activation (VvPS-TTb). The protective efficacy of conjugated and unconjugated formulations of VvPS and that of protein carriers were evaluated in a mouse septicemia model. Eighty percent of mice actively immunized with VvPS-TTa vaccine survived challenge with carbotype 1 V. vulnificus, while VvPS-cytolysin and VvPS-elastase conjugates conferred 44 and 40% protection, respectively. Control mice immunized with VvPS, cytolysin, or elastase alone, or saline only, showed 70 to 100% mortality. VvPS-TTa vaccine is nontoxic, immunogenic, and protective in mice.

Immunological properties of the cell surface haemagglutinins (sHAs) of Helicobacter pylori strain NCTC 11637.

Rabbits were immunised with stage 1 and stage 2 soluble haemagglutinins (sHA) of Helicobacter pylori strain NCTC 11637 and with rabbit erythrocytes coated with stage 1 sHA. After adsorption of stage 1 sHA on erythrocytes, SDS-PAGE analysis showed that 4 major protein bands were removed from the preparation. The anti-sHA coated erythrocyte serum had the highest HA inhibition titre of 16. Crossed immunoelectrophoresis of the stage 1 sHA, against stage 1 and 2 antisera showed multiple precipitin arcs; however, the anti-sHA coated erythrocyte serum produced only two arcs. One arc produced by the anti-stage 2 serum was absent with the anti-stage 1 serum. This arc could have been produced against a 20 kDa polypeptide which was absent in the stage 1 sHA. The other arc was stronger when compared with that produced by anti-stage 1 serum. These two arcs corresponded to the two arcs produced by the anti-sHA coated erythrocyte serum, which had the highest inhibition titre. The two arcs were markedly reduced in crossed immunoelectrophoresis with an adsorbed stage 1 sHA preparation, which indicates that these arcs were produced against the sHAs.

Inhibition of Staphylococcus adherence to biomaterials by extracellular slime of S. epidermidis RP12.

Adherence of selected strains of coagulase-negative staphylococci to various biomaterials, and the inhibition of their adherence by extracellular slime obtained from the RP12 strain of Staphylococcus epidermidis were studied in vitro. S. epidermidis RP12 adhered considerably more to polymethylmethacrylate (PMMA) discs than did the SP2 strain of S. hominis and the SE-360 strain of S. hyicus. Strain RP12 was less adherent to titanium alloy, ultrahigh molecular weight polyethylene (UHMWPE), and Teflon discs than to PMMA discs. Exposure of PMMA discs to extracellular slime extracted from strain RP12 greatly reduced adherence of strain RP12, SP2, SE-360, and S. epidermidis RP-62A. The active component(s) was present in the > 10 kD mol wt fraction obtained by Amicon YM10 ultrafiltration of crude slime; heat treatment of the fraction did not affect its inhibitory activity. When the bacteria and RP12 slime fractions were added simultaneously to the PMMA discs, the > 10 kD mol wt fraction of slime competitively inhibited adherence of strain RP12 to PMMA discs; in contrast, the < 10 kD mol wt fraction enhanced adherence of strain RP12 to PMMA discs.

The glycocalyx, biofilm, microbes, and resistant infection.

Biomaterial implants, traumatized tissues and bone are susceptible to immediate and delayed infections because microbes preferentially adhere to "inert biomaterials" or to damaged tissue surfaces. This type of infection is resistant to antibiotic therapy and most often requires removal of the prosthesis or infected tissue. This article discusses glycocalyx, biofilm, microbes, and resistant infection in prosthesis or infected tissue.

Isolation of a sialic acid-specific surface haemagglutinin of Helicobacter pylori strain NCTC 11637.

A deionized water extract of Helicobacter pylori NCTC 11637 contained haemagglutinin activity that was (i) soluble (i.e., not associated with particulate material sedimented by centrifugation at 100,000 x g for 1 h), (ii) stable to lyophilization, (iii) heat-labile, (iv) chymotrypsin-sensitive, (v) inhibited by fetuin, orosomucoid, and NANLac, but not by asialofetuin and (vi) inactive against guinea pig erythrocytes incubated with Clostridium perfringens neuraminidase, but active against untreated guinea pig erythrocytes. The data support the idea that the haemagglutinin is a protein which recognizes the alpha-(2-3) structure of sialylated glycoconjugates. Fractionation of the extract by isoelectric focusing and by gel filtration with Sephacryl S-400 indicated that the haemagglutinin has a pI of 3.7 and consist of high molecular-weight-protein aggregates. SDS-PAGE analysis of the preparation purified by gel filtration showed 3 protein bands at ca. 64 kD, 56 kD and 20 kD. Electron microscopy of H. pylori incubated with gold-labelled fetuin indicated that the haemagglutinin was associated with loosely adherent material on the bacterial surface, and that the purified haemagglutinin did not reveal a fimbrial structure. The ability to bind to sialoglycoconjugates on the erythrocyte membrane suggests that the haemagglutinin may be an important colonization factor enabling H. pylori to bind to similar saccharide structures on epithelial cells.

Effects of stonefish (Synanceia trachynis) venom on murine and frog neuromuscular junctions.

The neuromuscular toxicity of stonefish (Synanceia trachynis) venom was characterized by electrophysiological and electron microscopic examination of isolated murine and frog nerve-skeletal muscle preparations exposed to various concentrations of venom. Low concentrations of venom (2.5-10 micrograms/ml) acted presynaptically by causing release and depletion of neurotransmitter from the nerve terminal. The response was Na+ channel-independent (resistant to tetrodotoxin), required the presence of either Ca2+ or Mg2+, and was observed with botulinum neurotoxin-paralyzed nerve-muscle preparations. Higher concentrations of venom (100-300 micrograms/ml) acted postsynaptically and presynaptically. They caused irreversible depolarization of muscle cells and microscopically observable muscle and nerve damage. We conclude that the previously observed neuromuscular toxicity of stonefish venom is a consequence of the venom's dose-dependent, presynaptic and postsynaptic actions at the myoneural junction.

Detection of a cytolytic toxin in the venom of the stonefish (Synanceia trachynis).

The venom of the stonefish, Synanceia trachynis, contains a cytolytic toxin which is antigenic and ammonium sulfate-precipitable, and has a pI of ca 5.7 and an Mr of ca 158,000. The toxin is a potent but narrow-spectrum cytolysin which is lytic in vitro for rabbit, dog, rat, and guinea pig erythrocytes, in that order, but is largely or completely inactive against sheep, cow, human, monkey, mouse, goat, horse, burro and cat erythrocytes. Fractionation of the venom by molecular sieve fast protein liquid chromatography and isoelectric focusing did not separate the haemolytic activity from the venom's lethal and vascular permeability-increasing activities. Also, the three activities were destroyed by heat, proteases, Congo red, potassium permanganate and stonefish antivenoms. The results suggest that the haemolytic, lethal and vascular permeability-increasing activities of stonefish venom are properties of the same molecule, a previously unrecognized, membrane-damaging protein toxin.

Cloning and expression of the damselysin gene from Vibrio damsela.

The gene encoding damselysin, an extracellular cytolysin produced by virulent Vibrio damsela strains, was cloned and expressed in Escherichia coli. DNA sequences homologous to that of the cloned gene were detected in hemolytic strains of V. damsela but not in other hemolytic Vibrio species.

Detection of Vibrio vulnificus cytolysin in V. vulnificus-infected mice.

Enzyme-linked immunosorbent assays using polyclonal and monoclonal antibodies specific for V. vulnificus cytolysin detected the toxin in an extract of skin lesions and in serum from mice showing local and systemic V. vulnificus disease after subcutaneous injection of the bacterium. The cytolysin also was detected in skin lesions by an indirect immunofluorescence procedure using polyclonal and monoclonal antibodies. Our findings provide direct evidence that the cytolysin is produced in vivo during the development of the disease process, and this observation is consistent with the hypothesis that the toxin is involved in the pathogenesis of V. vulnificus disease.

Phospholipase D activity of Vibrio damsela cytolysin and its interaction with sheep erythrocytes.

Exposure of sheep erythrocytes to sublytic amounts of Vibrio damsela cytolysin markedly reduced their membrane sphingomyelin content and their sensitivity to lysis by the sphingomyelin-dependent cytolysins staphylococcal sphingomyelinase C (beta-toxin) and helianthin. The toxin was found to be a phospholipase D active against sphingomyelin.

Purification and characterization of an elastolytic protease of Vibrio vulnificus.

Large amounts of a highly purified, extracellular elastolytic protease of Vibrio vulnificus were obtained by sequential ammonium sulphate precipitation and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The protease had an Mr of about 50,500 (estimated by SDS-PAGE), a pI of 5.7, and a temperature optimum range of 55 to 60 degrees C. The pH optimum and the results of inactivation studies suggested that the enzyme was a neutral metalloprotease. The protease had about 429 amino acid residues, and the first 20 amino-terminal amino acid residues were Ala-Gln-Ala-Asn-Gly-Thr-Gly-Pro-Gly-Gly-Asn-Ser-Lys-Thr-Gly-Arg-Tyr-Glu- Phe-Gly . The purified protease was toxic for mice (about 1.5 mg kg-1 and 4.5 mg kg-1, intraperitoneal and intravenous LD50 values, respectively), and subcutaneous injection of the enzyme elicited rapid and extensive dermonecrosis.

Mouse skin damage caused by cytolysin from Vibrio vulnificus and by V. vulnificus infection.

Light and electron microscopy of mouse skin damage caused by intradermal infection with a virulent strain of Vibrio vulnificus and by a single intradermal injection of the cytolytic toxin produced by the bacterium revealed similar structural alterations. The epidermis was intact; however, the infection and toxin produced acute cellulitis characterized by extensive extracellular edema; disorganization of collagen bundles; large accumulations of cell debris and plasma proteins; damaged or necrotic fat cells, capillary endothelial cells, and muscle cells; and mild inflammatory cell infiltration. The virulent strain of V. vulnificus produced a capsule and was resistant to phagocytosis in vivo, whereas a weakly virulent strain of the bacterium did not produce a capsule and was readily phagocytized and digested. Factors that may be important in the pathogenesis of V. vulnificus wound infections include a capsule that inhibits phagocytosis and an extracellular cytolytic toxin that is responsible, at least in part, for the severe tissue damage characteristic of such wound infections.