Virulence of Yersinia ruckeri serotype I strains is associated with a heat sensitive factor (HSF) in cell extracts

Cell extracts of Yersinia ruckeri (serotype I) were examined by SDS-PAGE and Western blotting. An unusual band, termed heat-sensitive factor (HSF) was observed in extracts of virulent strains only. It is thought to be lipid in nature; no differences could be detected in the region of the band in protein profiles of virulent and avirulent strains. When trout were infected either by intraperitoneal injection or bath immersion, mortalities occurred only with HSF+ strains. The HSF appears to be an important virulence determinant of Y. ruckeri.     

Effect of Recovery Medium on the Isolation of Campylobacter Jejuni Before and After Heat Treatment

The effect of various concentrations of polymyxin B and Colistin in Skirrow’s or Butzler’s type media, respectively, on the recovery rate of 13 strains of G. jejuni before and after heat treatment was studied. Prior to heating, a Butz-ler’s type medium containing 40 I.U. per ml Colistin was inhibitory for certain strains of C. jejuni. After heating at 48° G for 30 min, media containing 2.5 I.U. per ml polymyxin B or 10 I.U. per ml Colistin were not inhibitory for heat-injured cells. When the concentrations of polymyxin B were increased to 5.0 I.U. per ml or those of Colistin to 20 or 40 I.U. per ml in the selective media, the means of the differences of log cell counts between non-selective Brucella blood agar and the selective media was statistically significant (P < 0.05). The mean D-value of G. jejuni strains in Brucella broth at 48° G was 18.4±5.4 min.     

Biotyping of Propionibacterium acnes isolated from normal human facial skin

Biochemical and serological characteristics of 128 strains of Propionibacterium acnes isolated from the facial skin of healthy Japanese volunteers were compared with the three standard strains of the American Type Culture Collection, ATCC 6919, 11827, and 11828. Accordingly, the isolated strains of P. acnes were classified into five biotypes (B1 to B5) on the basis of fermentation tests of ribose, erythritol, and sorbitol. Two serotypes were distinguished by the agglutination test. P. acnes belonging to serotype I had galactose as a cell wall sugar, whereas those of serotype II lacked galactose. The strains of serotype I were distributed among all five biotypes (B1 to B5); however, those of serotype II consisted only of one biotype (B2). A term "sero-biotype" was introduced to differentiate and carefully classify the isolates. The predominant sero-biotypes differed with the individual and region of the facial skin. In general, strains of sero-biotype IB1, IB3, IB4, and IIB2 were more frequently isolated than those of sero-biotype IB2 and IB5. Thus, for routine assay work, serotyping of P. acnes as based on erythritol and sorbitol fermentation is both practical and applicable.     

Effect of inorganic phosphorus on the biosynthesis of polymyxin B]

A synthetic medium containing optimal levels of the sources of carbon, nitrogen and inorganic phosphorus and providing satisfactory yields of polymyxin B was developed for 2 strains of Bac. polymyxa 933 and VK-153. The consumption of phosphorus in the medium by the strains and the antibiotic biosynthesis levels depended on the form of phosphorus added to the medium. Optimal biosynthesis of polymyxin B was observed at lower concentration levels of soluble soluble phosphorus in the medium than the bacterial growth.     

Enterotoxin-producing strains of Bacillus thuringiensis isolated from food

P.H. DAMGAARD, H.D. LARSEN, B.M. HANSEN, J. BRESCIANI AND K. JØRGENSEN. 1996. Strains of Bacillus thuringiensis were isolated from various food items (pasta, pitta bread and milk) and were found to belong to either H-serotype kurstaki or neoleonensis. The strains were bioassayed against Pieris brassicae and insecticidal activity of strains was found to correspond to the presence of the cry1.A -gene. All strains, except one, were found to express cytotoxic effects on Vero cells as an indicator of enterotoxin activity. Further, the B. thuringiensis strains HD-1 (serotype kurstuki ), NB-125 (serotype tenebrionis ) and HD-567 (serotype isruelensis ) which are used commercially for insect pest management, were also found to have cytotoxic effects on Vero cells.     

Metachromasy of peripheral nerve collagen on polyacrylamide gels stained with Coomassie brilliant blue R-250.

Endoneurial collagen stains metachromatically with Coomassie brilliant blue R-250 (C.I. 42660) when peripheral nerve proteins are solubilized with urea and SDS and then subjected to SDS-polyacrylamide gel electrophoresis. The metachromasy is reproducible under different fixing and staining conditions, but was exhibited only by Coomassie blue R-250 of the four triphenylmethane dyes tested. A method is presented for measurement of the degree of metachromasy on SDS gels and the detection of collagen in homogenates of whole tissue.     

Wall-associated protein antigens of Streptococcus mutans.

When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29 000, while B had a molecular weight of 190 000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

Variable cross-reactivity of Pseudomonas aeruginosa lipopolysaccharide-code-specific monoclonal antibodies and its possible relationship with serotype.

The core region of Pseudomonas aeruginosa lipopolysaccharide (LPS) was analysed by four LPS-core-specific human monoclonal antibodies (mAbs; FK-2E7, MH-4H7, OM-1D6 and NM-3G8). Reactivity of these mAbs to about 180 P. aeruginosa strains was tested. FK-2E7 bound to strains of Homma serotype E and I at a frequency of about 90%, to strains of serotype M at about 50%, and to strains of serotype A and G at about 30%. MH-4H7 bound to P. aeruginosa strains of serotype A, F, G, H, K and M at a high frequency (45-87%), but did not bind to any strains of serotype B, C, E and I. OM-1D6 and NM-3G8 bound to P. aeruginosa strains in a nearly serotype-specific manner. OM-1D6 reacted with all strains of serotype G so far tested, and a few strains of serotype M. Furthermore, L-rhamnose in the LPS core of serotype G was an immunodominant sugar recognized by OM-1D6 as an epitope. NM-3G8 bound to only a few strains of serotype B and M. The variable reactivity of these mAbs suggests that antigenic heterogeneity of the LPS core is somewhat related with (O-polysaccharide-based) serotype. Among these mAbs, MH-4H7 and OM-1D6 showed a high level of protective activity against P. aeruginosa in an experimental infection model using normal mice. In vivo protective activity was shown to be closely related to in vitro binding activity to whole cells as determined by agglutination and flow cytometry, but not ELISA.     

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system.

This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri. A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogeneicity of Y. ruckeri for fish.     

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system

Abstract: This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri . A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogenicity of Y. ruckeri for fish.     

Glycine-containing selective medium for isolation of Legionellaceae from environmental specimens.

Glycine, at a final concentration of 0.3%, has been shown to be an excellent selective agent for the isolation of Legionellaceae. Stock cultures of Legionella pneumophila were not inhibited on buffered charcoal-yeast extract agar containing the amino acid. Among the other Legionellaceae tested, only one of two strains of L. dumoffii and two of six strains of L. micdadei were appreciably inhibited. This medium permitted the isolation of L. pneumophila from environmental specimens with marked inhibition of many non-Legionellaceae bacteria. The selectivity of the medium was subsequently improved by the incorporation of vancomycin (5 microgram/ml) and polymyxin B (100 U/ml). This selective medium, glycine-vancomycin-polymyxin B agar, should facilitate the recovery of Legionellaceae from environmental sources.     

Serology of Neisseria gonorrhoeae. Classification by co-agglutination

The co-agglutination (COA) method has been adapted for serological classification of Neisseria gonorrhoeae. COA reagents were prepared with selectively absorbed rabbit hyperimmune antibodies against gonoccal (GC) major outer membrane protein (MOMP) serotype strains. Using these reagents, the 16 MOMP reference strains could be referred to at least three antigen classes, tentatively named W, J and M. The GC antigens of class W were divided into three groups I, II and III, and they were in part sensitive to pronase. The antigens of class J reflected strain specific or serotype reactions, some sensitive and others resistant to proteolytic enzymes. The antigens of class M were sensitive to periodate and resistant to pronase. Strains used in serological studies by other authors were tested. The properties of class W correlated well with those of the so-called micro-immunofluorescence and immunotype systems, and class M with those of the so-called endotoxin and acid polysaccharide systems. Strains from three different laboratories could all be grouped by class W and M reagents. Identical strains obtained independently from different laboratories gave very similar reaction patterns with the reagents available. Repeated GC-isolates from patients infected with beta-lactamase producing strains showed stable reactions with class W and J reagents, while there was a time-related variation of the class M pattern. We have found that the COA method is rapid, easy and reproducible in the serological classification of Neisseria gonorrhoeae and all the 117 GC-strains tested could be classified.     

Immunization of salmonids against Yersinia ruckeri: significance of humoral immunity and cross protection between serotypes

Brook trout (Salvelinus fontinalis) were immunized with bacterins containing either Serotype 1 or 2 isolates of Yersinia ruckeri to determine the relative degree of cross-protection afforded when the fish were challenged with the homologous or heterologous serotype. While fish immunized with pH-lysed bacterins produced highly specific agglutinins that did not cross-react with antigens derived from a heterologous serotype of Y. ruckeri all fish were protected against experimental challenge, regardless of which serotype was used for bacterin production and experimental challenge. Other experiments indicated that brook trout injected intraperitoneally with highly specific antibodies could not be passively immunized against experimental challenge.     

Distribution of restriction endonucleases among some entomopathogenic strains of Bacillus sphaericus

The restriction enzyme Bsp TI, an isoschizomer of Hae III (recognition site GGCC), has been detected in eight strains of serotype 5a5b and two serotype 3 strains of the entomopathogenic bacterium Bacillus sphaericus . Strains from other serotypes contained the enzymes Bsp TII and Bsp TIII, which digested pBR322 DNA into similar banding patterns after agarose gel electrophoresis but differed in their susceptibility to methylation of the substrate. Strains from serotypes 9, 25 and 26a26b were lacking in restriction enzyme activity. There was little correlation between phage typing and restriction enzyme activity, suggesting that restriction and modification are not responsible for phage specificity among entomopathogenic B. sphaericus strains.     

PURIFICATION AND PROPERTIES OF THE MUCUS OF EUGLENA GRACILIS (EUGLENOPHYCEAE)1

Anionic groups were demonstrated in the mucus of Euglena gracilis Klebs var. bacillaris Cori by histochemical staining with alcian blue or diaminobenzidine tetrahydrochloride and were quantified during the growth cycle with an alcian blue dye-binding assay. Mucus in the culture increased during growth and became high when the culture entered the stationary phase. Cultures were grown under conditions which uniformly labeled all sulfur containing compounds with ³⁵S. A purification scheme was devised using 0.15 M NaCl and 0.10 M EDTA at pH 8 (Marmur's solution) to separate the mucus from the cells without cell breakage. The isolated purified mucus was fractionated with sodium dodecyl sulfate (SDS) at 100° C into soluble and insoluble components. The soluble fraction was separated by SDS polyacrylamide gel electrophoresis into 18 polypeptide bands ranging from 22 to 320 kdaltons that stained with Coomassie blue; 16 of these bands also stained for carbohydrates using periodic acid-Schiffs reagent, indicating their glycoprotein nature. On hydrolysis, the SDS soluble fraction yielded xylose, fucose, rhamnose, and hexose. The SDS insoluble fraction contained no ³⁵S label, and, therefore, presumably no protein or bound sulfate; this gelatinous material does not contain the same sugar residues as the glycoproteins in the SDS soluble fraction. Its staining properties with alcian blue and its resistance to hydrolysis suggested the presence of uronic acids. Comparison with other Euglena fractions showed that bands comigrating with the mucus glycoproteins were not detectable in the fractions containing the whole cells or the culture medium. Although the mucus of Euglena yielded appreciable sulfate during mild acid treatment, most if not all of this sulfate appears to have come from the oxidation of reduced sulfur rather than from the hydrolysis of covalently bound sulfate. An infrared spectrum of the mucus showed only minor peaks in the correct regions for the S-O linkage. Thus, the mucus of Euglena is composed of glycoproteins and polysaccharides which contain little or no ester sulfate.     

Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon (Oncorhynchus kisutch) with endemic A. salmonicida and from cultured lake trout (Salvelinus namaycush) were tested by PCR-DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR-DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR-DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens, Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR-DGGE detection of this bacterium from mucus was possible. PCR-DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus.     

Fractionation of individual, biologically active factor VIII multimers

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.     

A simple infrared spectroscopic method for the measurement of expired 13CO2.

A simple method for determination of proteins, initially solubilized in Tris buffer containing sodium dodecyl sulfate (SDS), mercaptoethanol, and sucrose, is described. This method is based on protein and Coomassie brilliant blue G-250 binding but it involves the removal of excess SDS by precipitation with 100 mm potassium phosphate buffer, pH 7.4 to 7.5, prior to protein determination. It has been established that the precipitation of excess SDS does not lead to the removal of the solubilized proteins. Therefore the method is applicable to both water-soluble and water-insoluble protein samples.